In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells

ABSTRACT

The present invention relates to a high efficiency method of expressing immunoglobulin molecules in eukaryotic cells. The invention is further drawn to a method of producing immunoglobulin heavy and light chain libraries, particularly using the trimolecular recombination method, for expression in eukaryotic cells. The invention further provides methods of selecting and screening for antigen-specific immunoglobulin molecules, and antigen-specific fragments thereof. The invention also provides kits for producing, screening and selecting antigen-specific immunoglobulin molecules. Finally, the invention provides immunoglobulin molecules, and antigen-specific fragments thereof, produced by the methods provided herein.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional ApplicationNo. 60/249,268, filed Nov. 17, 2000, U.S. Provisional Application No.60/262,067, filed Jan. 18, 2001, U.S. Provisional Application No.60/271,424, filed Feb. 27, 2001, and U.S. Provisional Application No.60/298,087, filed Jun. 15, 2001, all of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to a high efficiency method ofexpressing immunoglobulin molecules in eukaryotic cells, a method ofproducing immunoglobulin heavy and light chain libraries for expressionin eukaryotic cells, methods of isolating immunoglobulins which bindspecific antigens, and immunoglobulins produced by any of these methods.

[0004] 2. Related Art

[0005] Immunoglobulin Production

[0006] Antibodies of defined specificity are being employed in anincreasing number of diverse therapeutic applications.

[0007] Defined antibodies directed against self antigens are ofparticular value for in vivo therapeutic and diagnostic purposes. Manyrodent monoclonal antibodies have been isolated using hybridomatechnology and utilized for in vivo therapeutic and diagnostic purposesin humans. For example, an early application of these mouse monoclonalantibodies was as targeting agents to kill or image tumors (F. H. Delandand D. M. Goldenberg 1982 in ‘Radionuclide Imaging’ ed. D. E. Kuhlpp289-297, Pergamon, Paris; R. Levy and R. A. Miller Ann. Rev. Med.1983, 34 pp107-116). However, the use of such antibodies in vivo canlead to problems. The foreign immunoglobulins can elicit ananti-immunoglobulin response which can interfere with therapy (R. A.Miller et al, 1983 Blood 62 988-995) or cause allergic or immune complexhypersensitivity (B. Ratner, 1943, Allergy, Anaphylaxis andImmunotherapy Williams and Wilkins, Baltimore). Accordingly, it isespecially important for such applications to develop antibodies thatare not themselves immunogenic in host, for example, to developantibodies against human antigens that are not themselves immunogenic inhumans.

[0008] It is a demanding task to isolate an antibody fragment withspecificity against self antigen. Animals do not normally produceantibodies to self antigens, a phenomenon called tolerance (Nossal, G.J. Science 245:147-153 (1989)). In general, vaccination with a selfantigen does not result in production of circulating antibodies. It istherefore difficult to raise antibodies to self antigens.

[0009] Previously, three general strategies have been employed toproduce immunoglobulin molecules which specifically recognize “self”antigens. In one approach, rodent antibody sequences have been convertedinto human antibody sequences, by grafting the specializedcomplementarity-determining regions (CDR) that comprise theantigen-binding site of a selected rodent monoclonal antibody onto theframework regions of a human antibody (Winter, et al., United KingdomPatent No. GB2188638B (1987); Reichmann. L., et al. Nature (London)332:323-327 (1988); Foote, J., and Winter, G. J. Mol. Biol. 224:487-499(1992)). In this approach, which has been termed antibody humanization,the three CDR loops of each rodent immunoglobulin heavy and light chainare grafted into homologous positions of the four framework regions of acorresponding human immunoglobulin chain. Because some of the frameworkresidues also contribute to antibody affinity, the structure must, ingeneral, be further refined by additional framework substitutions toenhance affinity. This can be a laborious and costly process.

[0010] More recently, transgenic mice have been generated that expresshuman immunoglobulin sequences (Mendez, M. J., et al., Nat. Genet.15:146-156 (1997)). While this strategy has the potential to accelerateselection of human antibodies, it shares with the antibody humanizationapproach the limitation that antibodies are selected from the availablemouse repertoire which has been shaped by proteins encoded in the mousegenome rather than the human genome. This could bias the epitopespecificity of antibodies selected in response to a specific antigen.For example, immunization of mice with a human protein for which a mousehomolog exists might be expected to result predominantly in antibodiesspecific for those epitopes that are different in humans and mice. Thesemay, however, not be the optimal target epitopes.

[0011] An alternative approach, which does not suffer this samelimitation, is to screen recombinant human antibody fragments displayedon bacteriophage (Vaughan, T. J., et al., Nat. Biotechnol. 14:309-314(1996); Barbas, C. F., III Nat. Med. 1:837-839 (1995); Kay, B. K., etal. (eds.) “Phage Display of Peptides and Proteins” Academic Press(1996)) In phage display methods, functional immunoglobulin domains aredisplayed on the surface of a phage particle which carriespolynucleotide sequences encoding them. In typical phage displaymethods, immunoglobulin fragments, e.g., Fab, Fv or disulfide stabilizedFv immunoglobulin domains are displayed as fusion proteins, i.e., fusedto a phage surface protein. Examples of phage display methods that canbe used to make the antibodies include those disclosed in Brinkman U. etal. (1995) J. Immunol. Methods 182:41-50; Ames, R. S. et al. (1995) J.Immunol. Methods 184:177-186; Kettleborough, C. A. et al. (1994) Eur. J.Immunol. 24:952-958; Persic, L. et al. (1997) Gene 187 9-18; Burton, D.R. et al. (1994) Advances in Immunology 57:191-280; PCT/GB91/01134; WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426, 5,223,409,5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698,5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743 (saidreferences incorporated by reference in their entireties).

[0012] Since phage display methods normally only result in theexpression of an antigen-binding fragment of an immunoglobulin molecule,after phage selection, the immunoglobulin coding regions from the phagemust be isolated and re-cloned to generate whole antibodies, includinghuman antibodies, or any other desired antigen binding fragment, andexpressed in any desired host including mammalian cells, insect cells,plant cells, yeast, and bacteria. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in WO92/22324; Mullinax, R. L. et al., BioTechniques 12(6):864-869 (1992);and Sawai, H. et al., AJRI 34:26-34 (1995); and Better, M. et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

[0013] Immunoglobulin libraries constructed in bacteriophage may derivefrom antibody producing cells of naive or specifically immunizedindividuals and could, in principle, include new and diverse pairings ofhuman immunoglobulin heavy and light chains. Although this strategy doesnot suffer from an intrinsic repertoire limitation, it requires thatcomplementarity determining regions (CDRs) of the expressedimmunoglobulin fragment be synthesized and fold properly in bacterialcells. Many antigen binding regions, however, are difficult to assemblecorrectly as a fusion protein in bacterial cells. In addition, theprotein will not undergo normal eukaryotic post-translationalmodifications. As a result, this method imposes a different selectivefilter on the antibody specificities that can be obtained.

[0014] There is a need, therefore, for an alternative method to identifyimmunoglobulin molecules, and antigen-specific fragments thereof, froman unbiased immunoglobulin repertoire that can be synthesized, properlyglycosylated and correctly assembled in eukaryotic cells.

[0015] Eukaryotic Expression Libraries. A basic tool in the field ofmolecular biology is the conversion of poly(A)⁺ mRNA to double-stranded(ds) cDNA, which then can be inserted into a cloning vector andexpressed in an appropriate host cell. A method common to many cDNAcloning strategies involves the construction of a “cDNA library” whichis a collection of cDNA clones derived from the poly(A)⁺ mRNA derivedfrom a cell of the organism of interest. For example, in order toisolate cDNAs which express immunoglobulin genes, a cDNA library mightbe prepared from pre B cells, B cells, or plasma cells. Methods ofconstructing cDNA libraries in different expression vectors, includingfilamentous bacteriophage, bacteriophage lambda, cosmids, andplasmidvectors, are known. Some commonly used methods are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2dEdition, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor,N.Y. (1990).

[0016] Many different methods of isolating target genes from cDNAlibraries have been utilized, with varying success. These include, forexample, the use of nucleic acid hybridization probes, which are labelednucleic acid fragments having sequences complementary to the DNAsequence of the target gene. When this method is applied to cDNA clonesin transformed bacterial hosts, colonies or plaques hybridizing stronglyto the probe are likely to contain the target DNA sequences.Hybridization methods, however, do not require, and do not measure,whether a particular cDNA clone is expressed. Alternative screeningmethods rely on expression in the bacterial host, for example, coloniesor plaques can be screened by immunoassay for binding to antibodiesraised against the protein of interest. Assays for expression inbacterial hosts are often impeded, however, because the protein may notbe sufficiently expressed in bacterial hosts, it may be expressed in thewrong conformation, and it may not be processed, and/or transported asit would in a eukaryotic system. Many of these problems have beenencountered in attempts to produce immunoglobulin molecules in bacterialhosts, as alluded to above.

[0017] Accordingly, use of mammalian expression libraries to isolatecDNAs encoding immunoglobulin molecules would offer several advantagesover bacterial libraries. For example, immunoglobulin molecules, andsubunits thereof, expressed in eukaryotic hosts should be functional andshould undergo any normal posttranslational modification. A proteinordinarily transported through the intracellular membrane system to thecell surface should undergo the complete transport process. Further, useof a eukaryotic system would make it possible to isolate polynucleotidesbased on functional expression of eukaryotic RNA or protein. Forexample, immunoglobulin molecules could be isolated based on theirspecificity for a given antigen.

[0018] With the exception of some recent lymphokine cDNAs isolated byexpression in COS cells (Wong, G. G., et al., Science 228:810-815(1985); Lee, F. et al., Proc. Natl. Acad. Sci. USA 83:2061-2065 (1986);Yokota, T., et al., Proc. Natl. Acad. Sci. USA 83:5894-5898 (1986);Yang, Y., et al., Cell 47:3-10 (1986)), few cDNAs have been isolatedfrom mammalian expression libraries. There appear to be two principalreasons for this: First, the existing technology (Okayama, H. et al.,Mol. Cell. Biol. 2:161-170 (1982)) for construction of large plasmidlibraries is difficult to master, and library size rarely approachesthat accessible by phage cloning techniques. (Huynh, T. et al., In: DNACloning Vol, I, A Practical Approach, Glover, D. M. (ed.), IRL Press,Oxford (1985), pp. 49-78). Second, the existing vectors are, with oneexception (Wong, G. G., et al., Science 228:810-815 (1985)), poorlyadapted for high level expression. Thus, expression in mammalian hostspreviously has been most frequently employed solely as a means ofverifying the identity of the protein encoded by a gene isolated by moretraditional cloning methods.

[0019] Poxvirus Vectors. Poxvirus vectors are used extensively asexpression vehicles for protein and antigen expression in eukaryoticcells. The ease of cloning and propagating vaccinia in a variety of hostcells has led to the widespread use of poxvirus vectors for expressionof foreign protein and as vaccine delivery vehicles (Moss, B., Science252:1662-7 (1991)).

[0020] Large DNA viruses are particularly useful expression vectors forthe study of cellular processes as they can express many differentproteins in their native form in a variety of cell lines. In addition,gene products expressed in recombinant vaccinia virus have been shown tobe efficiently processed and presented in association with MHC class Ifor stimulation of cytotoxic T cells. The gene of interest is normallycloned in a plasmid under the control of a promoter flanked by sequenceshomologous to a non-essential region in the virus and the cassette isintroduced into the genome via homologous recombination. A panoply ofvectors for expression, selection and detection have been devised toaccommodate a variety of cloning and expression strategies. However,homologous recombination is an ineffective means of making a recombinantvirus in situations requiring the generation of complex libraries orwhen the insert DNA is large. An alternative strategy for theconstruction of recombinant genomes relying on direct ligation of viralDNA “arms” to an insert and the subsequent rescue of infectious virushas been explored for the genomes of poxvirus (Merchlinsky, et al.,1992, Virology 190:522-526; Pfleiderer, et al., 1995, J. GeneralVirology 76:2957-2962; Scheiflinger, et al., 1992, Proc. Natl. Acad.Sci. USA 89:9977-9981), herpesvirus (Rixon, et al., 1990, J. GeneralVirology 71:2931-2939) and baculovirus (Ernst, et al., 1994, NucleicAcids Research 22:2855-2856).

[0021] Poxviruses are ubiquitous vectors for studies in eukaryotic cellsas they are easily constructed and engineered to express foreignproteins at high levels. The wide host range of the virus allows one tofaithfully express proteins in a variety of cell types. Direct cloningstrategies have been devised to extend the scope of applications forpoxvirus viral chimeras in which the recombinant genomes are constructedin vitro by direct ligation of DNA fragments to vaccinia “arms” andtransfection of the DNA mixture into cells infected with a helper virus(Merchlinsky, etal., 1992, Virology 190:522-526; Scheiflinger, et al.,1992, Proc. Natl. Acad. Sci. USA 89:9977-9981). This approach has beenused for high level expression of foreign proteins (Pfleiderer, et al.,1995, J. Gen. Virology 76:2957-2962) and to efficiently clone fragmentsas large as 26 kilobases in length (Merchlinsky, et al., 1992, Virology190:522-526).

[0022] Naked vaccinia virus DNA is not infectious because the viruscannot utilize cellular transcriptional machinery and relies on its ownproteins for the synthesis of viral RNA. Previously, temperaturesensitive conditional lethal (Merchlinsky, et al., 1992, Virology190:522-526) or non-homologous poxvirus fowlpox (Scheiflinger, et al.,1992, Proc. Natl. Acad. Sci. USA 89:9977-9981) have been utilized ashelper virus for packaging. An ideal helper virus will efficientlyfacilitate the production of infectious virus from input DNA, but willnot replicate in the host cell or recombine with the vaccinia DNAproducts. Fowlpox virus is a very useful helper virus for these reasons.It can enter mammalian cells and provide proteins required for thereplication of input vaccinia virus DNA. However, it does not recombinewith vaccinia DNA, and infectious fowlpox virions are not produced inmammalian cells. Therefore, it can be used at relatively highmultiplicity of infection (MOI).

[0023] Customarily, a foreign protein coding sequence is introduced intothe poxvirus genome by homologous recombination with infectious virus.In this traditional method, a previously isolated foreign DNA is clonedin a transfer plasmid behind a vaccinia promoter flanked by sequenceshomologous to a region in the poxvirus which is non-essential for viralreplication. The transfer plasmid is introduced into poxvirus-infectedcells to allow the transfer plasmid and poxvirus genome to recombine invivo via homologous recombination. As a result of the homologousrecombination, the foreign DNA is transferred to the viral genome.

[0024] Although traditional homologous recombination in poxviruses isuseful for expression of previously isolated foreign DNA in a poxvirus,the method is not conducive to the construction of libraries, since theoverwhelming majority of viruses recovered have not acquired a foreignDNA insert. Using traditional homologous recombination, therecombination efficiency is in the range of approximately 0.1% or less.Thus, the use of poxvirus vectors has been limited to subcloning ofpreviously isolated DNA molecules for the purposes of protein expressionand vaccine development.

[0025] Alternative methods using direct ligation vectors have beendeveloped to efficiently construct chimeric genomes in situations notreadily amenable for homologous recombination (Merchlinsky, M. etal.,1992, Virology 190:522-526; Scheiflinger, F. et al., 1992, Proc. Natl.Acad. Sci. USA. 89:9977-9981). In such protocols, the DNA from thegenome is digested, ligated to insert DNA in vitro, and transfected intocells infected with a helper virus (Merchlinsky, M. et al., 1992,Virology 190:522-526, Scheiflinger, F. et al., 1992, Proc. Natl. Acad.Sci. USA 89:9977-9981). In one protocol, the genome was digested at aunique NotI site and a DNA insert containing elements for selection ordetection of the chimeric genome was ligated to the genomic arms(Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA.89:9977-9981). This direct ligation method was described for theinsertion of foreign DNA into the vaccinia virus genome (Pfleiderer etal., 1995, J. General Virology 76:2957-2962).

[0026] Alternatively, the vaccinia WR genome was modified to producevNotI/tk by removing the NotI site in the HindIII F fragment andreintroducing a NotI site proximal to the thymidine kinase gene suchthat insertion of a sequence at this locus disrupts the thymidine kinasegene, allowing isolation of chimeric genomes via use of drug selection(Merchlinsky, M. et al., 1992, Virology 190:522-526). The directligation vector vNotI/tk allows one to efficiently clone and propagatepreviously isolated DNA inserts at least 26 kilobase pairs in length(Merchlinsky, M. et al., 1992, Virology, 190:522-526). Although largeDNA fragments are efficiently cloned into the genome, proteins encodedby the DNA insert will only be expressed at the low level correspondingto the thymidine kinase gene, a relatively weakly expressed early classgene in vaccinia. In addition, the DNA will be inserted in bothorientations at the NotI site, and therefore might not be expressed atall. Additionally, although the recombination efficiency using directligation is higher than that observed with traditional homologousrecombination, the resulting titer is relatively low.

[0027] Accordingly, poxvirus vectors were previously not used toidentify previously unknown genes of interest from a complex populationof clones, because a high efficiency, high titer-producing method ofcloning did not exist for pox viruses. More recently, however, thepresent inventor developed a method for generating recombinantpoxviruses using tri-molecular recombination. See Zauderer, WO00/028016, published May 18, 2000, which is incorporated herein byreference in its entirety.

[0028] Tri-molecular recombination is a novel, high efficiency, hightiter-producing method for producing recombinant poxviruses. Using thetri-molecular recombination method in vaccinia virus, the presentinventor has achieved recombination efficiencies of at least 90%, andtiters at least 2 orders of magnitude higher, than those obtained bydirect ligation. According to the tri-molecular recombination method, apoxvirus genome is cleaved to produce two nonhomologous fragments or“arms.” A transfer vector is produced which carries the heterologousinsert DNA flanked by regions of homology with the two poxvirus arms.The arms and the transfer vector are delivered into a recipient hostcell, allowing the three DNA molecules to recombine in vivo. As a resultof the recombination, a single poxvirus genome molecule is producedwhich comprises each of the two poxvirus arms and the insert DNA.

SUMMARY OF THE INVENTION

[0029] In accordance with one aspect of the present invention, there isprovided a method of identifying polynucleotides which encode anantigen-specific immunoglobulin molecule, or antigen-specific fragmentthereof, from libraries of polynucleotides expressed in eukaryoticcells.

[0030] Also provided is a method of identifying polynucleotides whichencode immunoglobulin molecules, or fragments thereof, which possessaltered effector function.

[0031] Also provided is a method of constructing libraries ofpolynucleotides encoding immunoglobulin subunit polypeptides ineukaryotic cells using virus vectors, where the libraries areconstructed by trimolecular recombination.

[0032] Further provided are methods of identifying host cells expressingantigen-specific immunoglobulin molecules, or antigen-specific fragmentsthereof on their surface by selecting and/or screening forantigen-induced cell death, antigen-induced signaling, orantigen-specific binding.

[0033] Also provided are methods of screening for soluble immunoglobulinmolecules, or antigen-specific fragments thereof, expressed fromeukaryotic host cells expressing libraries of polynucleotides encodingsoluble secreted immunoglobulin molecules, through antigen binding orthrough detection of an antigen- or organism-specific function of theimmunoglobulin molecule.

BRIEF DESCRIPTION OF THE FIGURES

[0034]FIG. 1. Selection for specific human antibody by antigen-inducedapoptosis.

[0035]FIG. 2A. Preparation of host cells which directly or indirectlyundergo cell death in response to antigen cross linking of surfaceimmunoglobulins.

[0036]FIG. 2B. Validation of modified CH33 host cells designed toundergo CTL-induced lysis or cell death in response to antigen crosslinking of surface immunoglobulins.

[0037]FIG. 3. Construction of pVHE

[0038]FIG. 4. Construction of pVKE and pVLE

[0039]FIG. 5. Selection for Specific Human Antibody by Antigen-dependentAdherence

[0040]FIG. 6. Schematic of the Tri-Molecular Recombination Method.

[0041]FIG. 7. Nucleotide Sequence of p7.5/tk and pEL/tk promoters. Thenucleotide sequence of the promoter and beginning of the thymidinekinase gene for v7.5/tk (SEQ ID NO: 140) and vEL/tk is shown (SEQ ID NO:142), and the corresponding amino acid sequence including the initiatorcodon and a portion of the open reading frame, designated wherein as SEQID NO: 141 and SEQ ID NO: 143, respectively.

[0042]FIG. 8. Construction of pVHEs.

[0043]FIG. 9. Attenuation of poxvirus-mediated cytopathic effects.

[0044]FIG. 10 Construction of scFv expression vectors.

[0045]FIG. 11 Construction of pVBE-X-G1.

[0046]FIG. 12A A modification in the nucleotide sequence of the p7.5/tk(SEQ ID NO:1) vaccinia transfer plasmid. A new vector, p7.5/ATG0/tk (SEQID NO:2), derived as described in the text from the p7.5/tk vacciniatransfer plasmid.

[0047]FIG. 12B A new vector, p7.5/ATG1/tk (SEQ ID NO:3) derived asdescribed in the text from the p7.5/tk vaccinia transfer plasmid.

[0048]FIG. 12C A new vector, p7.5/ATG2/tk (SEQ ID NO:4) derived asdescribed in the text from the p7.5/tk vaccinia transfer plasmid.

[0049]FIG. 12D A new vector, p7.5/ATG3/tk (SEQ ID NO:5) derived asdescribed in the text from the p7.5/tk vaccinia transfer plasmid.

[0050]FIG. 13 Construction of IgM-Fas fusion products.

[0051]FIG. 14. Expression of Igα and Igβ in the transfected COS7 andHeLaS3 cell lines. Total RNA was isolated from (A) COS7-Igαβ-1, (B)COS7-Igαβ-2, (C) HeLaS3-Igαβ-1 and (D) EBV-transformed human B cells,reverse transcribed into cDNA in the presence or absence of reversetranscriptase, then PCR amplified with the igα5′/igα3′ and igβ5′/igβ3′primer sets. PCR products were then analyzed on 0.8% agarose gels. Itshould be noted that human B cells exhibit alternative splicing for bothIgα and Igβ See, e.g., Hashimoto, S., et al., Mol. Immunol.32:651(1995).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] The present invention is broadly directed to methods ofidentifying and/or producing functional, antigen-specific immunoglobulinmolecules, or antigen-specific fragments (i.e., antigen-bindingfragments) thereof, in a eukaryotic system. In addition, the inventionis directed to methods of identifying polynucleotides which encode anantigen-specific immunoglobulin molecule, or an antigen-specificfragment thereof, from complex expression libraries of polynucleotidesencoding such immunoglobulin molecules or fragments, where the librariesare constructed and screened in eukaryotic host cells. Furtherembodiments include an isolated antigen-specific immunoglobulinmolecule, or antigen-specific fragment thereof, produced by any of theabove methods, and a kit allowing production of such isolatedimmunoglobulins.

[0053] A particularly preferred aspect of the present invention is theconstruction of complex immunoglobulin libraries in eukaryotic hostcells using poxvirus vectors constructed by trimolecular recombination.The ability to construct complex cDNA libraries in a pox virus basedvector and to select and/or screen for specific recombinants on thebasis of either antigen induced cell death, antigen induced signaling,or antigen-specific binding can be the basis for identification ofimmunoglobulins, particularly human immunoglobulins, with a variety ofwell-defined specificities in eukaryotic cells. It would overcome thebias imposed by selection of antibody repertoire in rodents or thelimitations of synthesis and assembly in phage or bacteria.

[0054] It is to be noted that the term “a” or “an” entity, refers to oneor more of that entity; for example, “an immunoglobulin molecule,” isunderstood to represent one or more immunoglobulin molecules. As such,the terms “a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

[0055] The term “eukaryote” or “eukaryotic organism” is intended toencompass all organisms in the animal, plant, and protist kingdoms,including protozoa, fungi, yeasts, green algae, single celled plants,multi celled plants, and all animals, both vertebrates andinvertebrates. The term does not encompass bacteria or viruses. A“eukaryotic cell” is intended to encompass a singular “eukaryotic cell”as well as plural “eukaryotic cells,” and comprises cells derived from aeukaryote.

[0056] The term “vertebrate” is intended to encompass a singular“vertebrate” as well as plural “vertebrates,” and comprises mammals andbirds, as well as fish, reptiles, and amphibians.

[0057] The term “mammal” is intended to encompass a singular “mammal”and plural “mammals,” and includes, but is not limited to humans;primates such as apes, monkeys, orangutans, and chimpanzees; canids suchas dogs and wolves; felids such as cats, lions, and tigers; equids suchas horses, donkeys, and zebras, food animals such as cows, pigs, andsheep; ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears. Preferably, the mammal is a humansubject.

[0058] The terms “tissue culture” or “cell culture” or “culture” or“culturing” refer to the maintenance or growth of plant or animal tissueor cells in vitro under conditions that allow preservation of cellarchitecture, preservation of cell function, further differentiation, orall three. “Primary tissue cells” are those taken directly from tissue,i.e., a population of cells of the same kind performing the samefunction in an organism. Treating such tissue cells with the proteolyticenzyme trypsin, for example, dissociates them into individual primarytissue cells that grow or maintain cell architecture when seeded ontoculture plates. Cell cultures arising from multiplication of primarycells in tissue culture are called “secondary cell cultures.” Mostsecondary cells divide a finite number of times and then die. A fewsecondary cells, however, may pass through this “crisis period,” afterwhich they are able to multiply indefinitely to form a continuous “cellline.” The liquid medium in which cells are cultured is referred toherein as “culture medium” or “culture media.” Culture medium into whichdesired molecules, e.g., immunoglobulin molecules, have been secretedduring culture of the cells therein is referred to herein as“conditioned medium.”

[0059] The term “polynucleotide” refers to any one or more nucleic acidsegments, or nucleic acid molecules, e.g., DNA or RNA fragments, presentin a nucleic acid or construct. A “polynucleotide encoding animmunoglobulin subunit polypeptide” refers to a polynucleotide whichcomprises the coding region for such a polypeptide. In addition, apolynucleotide may encode a regulatory element such as a promoter or atranscription terminator, or may encode a specific element of apolypeptide or protein, such as a secretory signal peptide or afunctional domain.

[0060] As used herein, the term “identify” refers to methods in whichdesired molecules, e.g., polynucleotides encoding immunoglobulinmolecules with a desired specificity or function, are differentiatedfrom a plurality or library of such molecules. Identification methodsinclude “selection” and “screening.” As used herein, “selection” methodsare those in which the desired molecules may be directly separated fromthe library. For example, in one selection method described herein, hostcells comprising the desired polynucleotides are directly separated fromthe host cells comprising the remainder of the library by undergoing alytic event and thereby being released from the substrate to which theremainder of the host cells are attached. As used herein, “screening”methods are those in which pools comprising the desired molecules aresubjected to an assay in which the desired molecule can be detected.Aliquots of the pools in which the molecule is detected are then dividedinto successively smaller pools which are likewise assayed, until a poolwhich is highly enriched from the desired molecule is achieved. Forexample, in one screening method described herein, pools of host cellscomprising library polynucleotides encoding immunoglobulin molecules areassayed for antigen binding through expression of a reporter molecule.

[0061] Immunoglobulins. As used herein, an “immunoglobulin molecule” isdefined as a complete, bi-molecular immunoglobulin, i.e., generallycomprising four “subunit polypeptides,” i.e., two identical heavy chainsand two identical light chains. In some instances, e.g., immunoglobulinmolecules derived from camelid species or engineered based on camelidimmunglobulins, a complete immunoglobulin molecule may consist of heavychains only, with no light chains. See, e.g., Hamers-Casterman et al.,Nature 363:446-448 (1993). Thus, by an “immunoglobulin subunitpolypeptide” is meant a single heavy chain polypeptide or a single lightchain polypeptide. Immunoglobulin molecules are also referred to as“antibodies,” and the terms are used interchangeably herein. An“isolated immunoglobulin” refers to an immunoglobulin molecule, or twoor more immunoglobulin molecules, which are substantially removed fromthe milieu of proteins and other substances, and which bind a specificantigen.

[0062] The heavy chain, which determines the “class” of theimmunoglobulin molecule, is the larger of the two subunit polypeptides,and comprises a variable region and a constant region. By “heavy chain”is meant either a full-length secreted heavy chain form, i.e., one thatis released from the cell, or a membrane bound heavy chain form, i.e.,comprising a membrane spanning domain and an intracellular domain. Themembrane spanning and intracellular domains can be thenaturally-occurring domains associated with a certain heavy chain, i.e.,the domain found on memory B-cells, or it may be a heterologous membranespanning and intracellular domain, e.g., from a different immunoglobulinclass or from a heterologous polypeptide, i.e., a non-immunoglobulinpolypeptide. As will become apparent, certain aspects of the presentinvention are preferably carried out using cell membrane-boundimmunoglobulin molecules, while other aspects are preferably carried outwith using secreted immunoglobulin molecules, i.e., those lacking themembrane spanning and intracellular domains. Immunoglobulin “classes”refer to the broad groups of immunoglobulins which serve differentfunctions in the host. For example, human immunoglobulins are dividedinto five classes, i.e., IgG, comprising a γ heavy chain, IgM,comprising a μ heavy chain, IgA, comprising an α heavy chain, IgE,comprising an ε heavy chain, and IgD, comprising a δ heavy chain.Certain classes of immunoglobulins are also further divided into“subclasses.” For example, in humans, there are four different IgGsubclasses, IgG1, IgG2, IgG3, and IgG4 comprising γ-1, γ-2, γ-3, and γ-4heavy chains, respectively, and two different IgA subclasses, IgA-1 andIgA-2, comprising α-1 and α-2 heavy chains, respectively. It is to benoted that the class and subclass designations of immunoglobulins varybetween animal species, and certain animal species may compriseadditional classes of immunoglobulins. For example, birds also produceIgY, which is found in egg yolk.

[0063] By “light chain” is meant the smaller immunoglobulin subunitwhich associates with the amino terminal region of a heavy chain. Aswith a heavy chain, a light chain comprises a variable region and aconstant region. There are two different kinds of light chains, κ and λ,and a pair of these can associate with a pair of any of the variousheavy chains to form an immunoglobulin molecule.

[0064] Immunoglobulin subunit polypeptides each comprise a constantregion and a variable region. In most species, the heavy chain variableregion, or V_(H) domain, and the light chain variable region, or V_(L)domain, combine to form a “complementarity determining region” or CDR,the portion of an immunoglobulin molecule which specifically recognizesan antigenic epitope. In camelid species, however, the heavy chainvariable region, referred to as V_(H)H, forms the entire CDR. The maindifferences between camelid V_(H)H variable regions and those derivedfrom conventional antibodies (V_(H)) include (a) more hydrophobic aminoacids in the light chain contact surface of V_(H) as compared to thecorresponding region in V_(H)H, (b) a longer CDR3 in V_(H)H, and (c) thefrequent occurrence of a disulfide bond between CDR1 and CDR3 in V_(H)H.Each complete immunoglobulin molecule comprises two identical CDRs. Alarge repertoire of variable regions associated with heavy and lightchain constant regions are produced upon differentiation ofantibody-producing cells in an animal through rearrangements of a seriesof germ line DNA segments which results in the formation of a gene whichencodes a given variable region. Further variations of heavy and lightchain variable regions take place through somatic mutations indifferentiated cells. The structure and in vivo formation ofimmunoglobulin molecules is well understood by those of ordinary skillin the art of immunology. Concise reviews of the generation ofimmunoglobulin diversity may be found, e.g., in Harlow and Lane,Antibodies, A Laboratory Manual Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1988) (hereinafter, “Harlow”); and Roitt, et al.,Immunology Gower Medical Publishing, Ltd., London (1985) (hereinafter,“Roitt”). Harlow and Roitt are incorporated herein by reference in theirentireties.

[0065] Immunoglobulins further have several effector functions mediatedby binding of effector molecules. For example, binding of the C1component of complement to an immunoglobulin activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, immunoglobulins bind to cells via the Fcregion, with an Fc receptor site on the antibody Fc region binding to anFc receptor (FcR) on a cell. There are a number of Fc receptors whichare specific for different classes of antibody, including, but notlimited to, IgG (gamma receptors), IgE (eta receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

[0066] Immunoglobulins of the present invention may be from any animalorigin including birds, fish, and mammals. Preferably, the antibodiesare of human, mouse, dog, cat, rabbit, goat, guinea pig, camel, llama,horse, or chicken origin. In a preferred aspect of the presentinvention, immunoglobulins are identified which specifically interactwith “self” antigens, e.g., human immunoglobulins which specificallybind human antigens.

[0067] As used herein, an “antigen-specific fragment” of animmunoglobulin molecule is any fragment or variant of an immunoglobulinmolecule which remains capable of binding an antigen. Antigen-specificfragments include, but are not limited to, Fab, Fab′ and F(ab′)₂, Fd,single-chain Fvs (scFv), single-chain immunoglobulins (e.g., wherein aheavy chain, or portion thereof, and light chain, or portion thereof,are fused), disulfide-linked Fvs (sdFv), diabodies, triabodies,tetrabodies, scFv minibodies, Fab minibodies, and dimeric scFv and anyother fragments comprising a V_(L) and a V_(H) domain in a conformationsuch that a specific CDR is formed. Antigen-specific fragments may alsocomprise a V_(H)H domain derived from a camelid antibody. The V_(H)H maybe engineered to include CDRs from other species, for example, fromhuman antibodies. Alternatively, a human-derived heavy chain V_(H)fragment may be engineered to resemble a single-chain camelid CDR, aprocess referred to as “camelization.” See, e.g., Davies J., andRiechmann, L., FEBS Letters 339:285-290 (1994), and Riechmann, L., andMuyldermans, S., J. Immunol. Meth. 231:25-38 (1999), both of which areincorporated herein by reference in their entireties.

[0068] Antigen-specific immunoglobulin fragments, including single-chainimmunoglobulins, may comprise the variable region(s) alone or incombination with the entire or partial of the following: a heavy chainconstant domain, or portion thereof, e.g., a CH1, CH2, CH3,transmembrane, and/or cytoplasmic domain, on the heavy chain, and alight chain constant domain, e.g., a C_(κ) or C_(λ) domain, or portionthereof on the light chain. Also included in the invention are anycombinations of variable region(s) and CH1, CH2, CH3, C_(κ), C_(λ),transmembrane and cytoplasmic domains.

[0069] As is known in the art, Fv comprises a VH domain and a VL domain,Fab comprises VH joined to CH1 and an L chain, a Fab minibody comprisesa fusion of CH3 domain to Fab, etc.

[0070] As is known in the art, scFv comprises VH joined to VL by apeptide linker, usually 15-20 residues in length, diabodies comprisescFv with a peptide linker about 5 residues in length, triabodiescomprise scFv with no peptide linker, tetrabodies comprise scFv withpeptide linker 1 residue in length, a scFv minibody comprises a fusionof CH3 domain to scFv, and dimeric scFv comprise a fusion of two scFvsin tandem using another peptide linker (reviewed in Chames and Baty,FEMS Microbiol. Letts. 189:1-8 (2000)). Preferably, an antigen-specificimmunoglobulin fragment includes both antigen binding domains, i.e.,V_(H) and V_(L). Other immunoglobulin fragments are well known in theart and disclosed in well-known reference materials such as thosedescribed herein.

[0071] In certain embodiments, the present invention is drawn to methodsto identify, i.e., select or alternatively screen for, polynucleotideswhich singly or collectively encode antigen-specific immunoglobulinmolecules, antigen-specific fragments thereof, or immunoglobulinmolecules or fragments with specific antigen-related functions. Inrelated embodiments, the present invention is drawn to isolatedimmunoglobulin molecules encoded by the polynucleotides identified bythese methods.

[0072] The preferred methods comprise a two-step screening and/orselection process. In the first step, a polynucleotide encoding a firstimmunoglobulin subunit, i.e., either a heavy chain or a light chain, isidentified from a library of polynucleotides encoding that subunit byintroducing the library into a population of eukaryotic host cells, andexpressing the immunoglobulin subunit in combination with one or morespecies of a second immunoglobulin subunit, where the secondimmunoglobulin subunit is not the same as the first immunoglobulinsubunit, i.e., if the first immunoglobulin subunit polypeptide is aheavy chain polypeptide, the second immunoglobulin subunit polypeptidewill be a light chain polypeptide.

[0073] Once one or more polynucleotides encoding one or more firstimmunoglobulin subunit are isolated from the library in the first step,a second immunoglobulin subunit is identified in the second step.Isolated polynucleotides encoding the isolated first immunoglobulinsubunit polypeptide(s) are transferred-into and expressed in host cellsin which a library of polynucleotides encoding the second immunoglobulinsubunit are expressed, thereby allowing identification of apolynucleotide encoding a second immunoglobulin subunit polypeptidewhich, when combined with the first immunoglobulin subunit identified inthe first step, forms a functional immunoglobulin molecule, or fragmentthereof, which recognizes a specific antigen and/or performs a specificfunction.

[0074] Where immunoglobulin fragments are composed of one polypeptide,i.e., a single-chain fragment or a fragment comprising a V_(H)H domain,and therefore encoded by one polynucleotide, preferred methods comprisea one-step screening and/or selection process. Polynucleotides encodinga single-chain fragment, comprising a heavy chain variable region and alight chain variable region, or comprising a V_(H)H region, areidentified from a library by introducing the library into host cellssuch as eukaryotic cells and recovering polynucleotides of said libraryfrom those host cells which encode immunoglobulin fragments.

[0075] In certain embodiments, particular immunoglobulin molecules areidentified through contacting the host cells expressing immunoglobulinmolecules on their surface to antigen, which allows for selection and/orscreening of antigen-binding cells in a number of different ways asdescribed below. In other embodiments, desired soluble secretedimmunoglobulin molecules are identified by assaying pools of conditionedmedia for desired functional characteristics of the immunoglobulinmolecule, e.g., virus neutralization.

[0076] Where the immunoglobulin molecules are bound to the host cellsurface, the first step comprises introducing into a population of hostcells capable of expressing the immunoglobulin molecule a first libraryof polynucleotides encoding a plurality of first immunoglobulin subunitpolypeptides through operable association with a transcriptional controlregion, introducing into the same host cells a second library ofpolynucleotides encoding, through operable association with atranscriptional control region, a plurality of second immunoglobulinsubunit polypeptides, permitting expression of immunoglobulin molecules,or antigen-specific fragments thereof, on the membrane surface of thehost cells, contacting the host cells with an antigen, and recoveringpolynucleotides derived from the first library from those host cellswhich bind the antigen.

[0077] Where the immunoglobulin molecules are fully secreted into thecell medium, the first step comprises introducing into a population ofhost cells capable of expressing the immunoglobulin molecule a firstlibrary of polynucleotides encoding a plurality of first immunoglobulinsubunit polypeptides through operable association with a transcriptionalcontrol region, introducing into the same host cells a second library ofpolynucleotides encoding, through operable association with atranscriptional control region, a plurality of second immunoglobulinsubunit polypeptides, permitting expression and secretion ofimmunoglobulin molecules, or antigen-specific fragments thereof, intothe cell medium, assaying aliquots of conditioned medium for desiredantigen-related antibody functions, and recovering polynucleotidesderived from the first library from those host cell pools grown inconditioned medium in which the desired function was observed.

[0078] As used herein, a “library” is a representative genus ofpolynucleotides, i.e., a group of polynucleotides related through, forexample, their origin from a single animal species, tissue type, organ,or cell type, where the library collectively comprises at least twodifferent species within a given genus of polynucleotides. A library ofpolynucleotides preferably comprises at least 10, 100, 10³, 10⁴, 10⁵,10⁶, 10⁷, 10⁸, or 10⁹ different species within a given genus ofpolynucleotides. More specifically, a library of the present inventionencodes a plurality of a certain immunoglobulin subunit polypeptide,i.e., either a heavy chain subunit polypeptide or a light chain subunitpolypeptide. In this context, a “library” of the present inventioncomprises polynucleotides of a common genus, the genus beingpolynucleotides encoding an immunoglobulin subunit polypeptide of acertain type and class e.g., a library might encode a human μ, γ-1, γ-2,γ-3, γ-4, α-1, α-2, ε, or δ heavy chain, or a human κ or λ light chain.Although each member of any one library of the present invention willencode the same heavy or light chain constant region, the library willcollectively comprise at least two, preferably at least 10, 100, 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different variable regions i.e., a“plurality” of variable regions associatedwiththe common constantregion.

[0079] In other embodiments, the library encodes a plurality ofimmunoglobulin single-chain fragments which comprise a variable region,such as a light chain variable region or a heavy chain variable region,and preferably comprises both a light chain variable region and a heavychain variable region. Optionally, such a library comprisespolynucleotides encoding an immunoglobulin subunit polypeptide of acertain type and class, or domains thereof.

[0080] In one aspect, the present invention encompasses methods toproduce libraries of polynucleotides encoding immunoglobulin subunits.Furthermore, the present invention encompasses libraries ofimmunoglobulin subunits constructed in eukaryotic expression vectorsaccording to the methods described herein. Such libraries are preferablyproduced in eukaryotic virus vectors, even more preferably in poxvirusvectors. Such methods and libraries are described herein.

[0081] By “recipient cell” or “host cell” or “cell” is meant a cell orpopulation of cells into which polynucleotide libraries of the presentinvention are introduced. A host cell of the present invention ispreferably a eukaryotic cell or cell line, preferably a plant, animal,vertebrate, mammalian, rodent, mouse, primate, or human cell or cellline. By “a population of host cells” is meant a group of cultured cellsinto which a “library” of the present invention can be introduced andexpressed. Any host cells which will support expression from a givenlibrary constructed in a given vector is intended. Suitable andpreferred host cells are disclosed herein. Furthermore, certain hostcells which are preferred for use with specific vectors and withspecific selection and/or screening schemes are disclosed herein.Although it is preferred that a population of host cells be amonoculture, i.e., where each cell in the population is of the same celltype, mixed cultures of cells are also contemplated. Host cells of thepresent invention may be adherent, i.e., host cells which grow attachedto a solid substrate, or, alternatively, the host cells may be insuspension. Host cells may be cells derived from primary tumors, cellsderived from metastatic tumors, primary cells, cells which have lostcontact inhibition, transformed primary cells, immortalized primarycells, cells which may undergo apoptosis, and cell lines derivedtherefrom.

[0082] As noted above, preferred methods to identify immunoglobulinmolecules comprise the introduction of a “first” library ofpolynucleotides into a population of host cells, as well as a “second”library of polynucleotides into the same population of host cells. Thefirst and second libraries are complementary, i.e., if the “first”library encodes immunoglobulin heavy chains, the “second” library willencode immunoglobulin light chains, thereby allowing assembly ofimmunoglobulin molecules, or antigen-specific fragments thereof, in thepopulation of host cells. Also, as noted above, another method toidentify immunoglobulins or immunoglobulin fragments comprisesintroduction of a single library of polynucleotides encodingsingle-chain fragments into a population of host cells. The descriptionof polynucleotide libraries, the composition of the polynucleotides inthe library, and the polypeptides encoded by the polynucleotidestherefore encompass both the polynucleotides which comprise the “firstlibrary” and the polynucleotides which comprise the “second library,”and the polypeptides encoded thereby. The libraries may be constructedin any suitable vectors, and both libraries may, but need not be,constructed in the same vector. Suitable and preferred vectors for thefirst and second libraries are disclosed infra.

[0083] Polynucleotides contained in libraries of the present inventionencode immunoglobulin subunit polypeptides through “operable associationwith a transcriptional control region.” One or more nucleic acidmolecules in a given polynucleotide are “operably associated” when theyare placed into a functional relationship. This relationship can bebetween a coding region for a polypeptide and a regulatory sequence(s)which are connected in such a way as to permit expression of the codingregion when the appropriate molecules (e.g., transcriptional activatorproteins, polymerases, etc.) are bound to the regulatory sequences(s).“Transcriptional control regions” include, but are not limited topromoters, enhancers, operators, and transcription termination signals,and are included with the polynucleotide to direct its transcription.For example, a promoter would be operably associated with a nucleic acidmolecule encoding an immunoglobulin subunit polypeptide if the promoterwas capable of effecting transcription of that nucleic acid molecule.Generally, “operably associated” means that the DNA sequences arecontiguous or closely connected in a polynucleotide. However, sometranscription control regions, e.g., enhancers, do not have to becontiguous.

[0084] By “control sequences” or “control regions” is meant DNAsequences necessary for the expression of an operably associated codingsequence in a particular host organism. The control sequences that aresuitable for prokaryotes, for example, include a promoter, optionally anoperator sequence, and a ribosome binding site. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhances.

[0085] A variety of transcriptional control regions are known to thoseskilled in the art. Preferred transcriptional control regions includethose which function in vertebrate cells, such as, but not limited to,promoter and enhancer sequences from poxviruses, adenoviruses,herpesviruses, e.g., human cytomegalovirus (preferably the intermediateearly promoter, preferably in conjunction with intron-A), simian virus40 (preferably the early promoter), retroviruses (such as Rous sarcomavirus), and picornaviruses (particularly an internal ribosome entrysite, or IRES, enhancer region, also referred to herein as a CITEsequence). Other preferred transcriptional control regions include thosederived from mammalian genes such as actin, heat shock protein, andbovine growth hormone, as well as other sequences capable of controllinggene expression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas inducible promoters (e.g., promoters inducible by tetracycline, andtemperature sensitive promoters). As will be discussed in more detailbelow, especially preferred are promoters capable of functioning in thecytoplasm of poxvirus-infected cells.

[0086] In certain preferred embodiments, each “immunoglobulin subunitpolypeptide,” i.e., either a “first immunoglobulin subunit polypeptide”or a “second immunoglobulin subunit polypeptide” comprises (i) a firstimmunoglobulin constant region selected from the group consisting of aheavy chain constant region, either a membrane bound form of a heavychain constant region or a fully secreted form of a heavy chain constantregion; and a light chain constant region, (ii) an immunoglobulinvariable region corresponding to the first constant region, i.e., if theimmunoglobulin constant region is a heavy chain constant region, theimmunoglobulin variable region preferably comprises a V_(H) region, andif the immunoglobulin constant region is a light chain constant region,the immunoglobulin variable region preferably comprises a V_(L) region,and (iii) a signal peptide capable of directing transport of theimmunoglobulin subunit polypeptide through the endoplasmic reticulum andthrough the host cell plasma membrane, either as a membrane-bound orfully secreted heavy chain, or a light chain associated with a heavychain. Accordingly, through the association of two identical heavychains and two identical light chains, either a surface immunoglobulinmolecule or a fully secreted immunoglobulin molecule is formed.

[0087] Also in certain preferred embodiments in the context of animmunoglobulin fragment, a single-chain fragment comprises animmunoglobulin variable region selected from the group consisting of aheavy chain variable region and a light chain variable region, andpreferably comprises both variable regions. If the immunoglobulinfragment comprises both a heavy chain variable region and a light chainvariable region, they may be directly joined (i.e., they have no peptideor other linker), or they may be joined by another means. If they arejoined by other means, they may be joined directly or by a disulfidebond formed during expression or by a peptide linker, as discussedbelow. Accordingly, through the association of the heavy chain variableregion and the light chain variable region, a CDR is formed.

[0088] The heavy chain variable region and light chain variable regionof one single-chain fragment may associate with one another or the heavychain variable region of one single-chain fragment may associate with alight chain variable region of another single-chain fragment, and viseversa, depending on the type of linker. In one embodiment, thesingle-chain fragment also comprises a constant region selected from thegroup consisting of a heavy chain constant region, or a domain thereof,and a light chain constant region, or a domain thereof. Two single-chainfragments may associate with one another via their constant regions.

[0089] As mentioned above, in certain embodiments, the polynucleotideencoding the light chain variable region and heavy chain variable regionof the single-chain fragment encode a linker. The single-chain fragmentmay comprise a single polypeptide with the sequence V_(H)-linker-V_(L)or V_(L)-linker-V_(H). In some embodiments, the linker is chosen topermit the heavy chain and light chain of a single polypeptide to bindtogether in their proper conformational orientation. See for example,Huston, J. S., et al, Methods in Enzym. 203:46-121 (1991). Thus, inthese embodiments, the linker should be able to span the 3.5 nm distancebetween its points of fusion to the variable domains without distortionof the native Fv conformation. In these embodiments, the amino acidresidues constituting the linker are such that it can span this distanceand should be 5 amino acids or longer. Single-chain fragments with alinker of 5 amino acids form are found in monomer and predominantlydimer form. Preferably, the linker should be at least about 10 or atleast about 15 residues in length. In other embodiments, the linkerlength is chosen to promote the formation of scFv tetramers(tetrabodies), and is 1 amino acid in length. In some embodiments, thevariable regions are directly linked (i.e., the single-chain fragmentcontains no peptide linker) to promote the formation of scFv trimers(triabodies). These variations are well known in the art. (See, forexample, Chames and Baty, FEMS Microbiol. Letts. 189:1-8 (2000). Thelinker should not be so long it causes steric interference with thecombining site. Thus, it preferably should be about 25 residues or lessin length.

[0090] The amino acids of the peptide linker are preferably selected sothat the linker is hydrophilic so it does not get buried into theantibody. The linker (Gly-Gly-Gly-Gly-Ser)₃ (SEQ ID NO:6) is a preferredlinker that is widely applicable to many antibodies as it providessufficient flexibility. Other linkers include Glu Ser Gly Arg Ser GlyGly Gly Gly Ser Gly Gly Gly Gly Ser (SEQ ID NO:7), Glu Gly Lys Ser SerGly Ser Gly Ser Glu Ser Lys Ser Thr (SEQ ID NO:8), Glu Gly Lys Ser SerGly Ser Gly Ser Glu Ser Lys Ser Thr Gln (SEQ ID NO:9), Glu Gly Lys SerSer Gly Ser Gly Ser Glu Ser Lys Val Asp (SEQ ID NO:10), Gly Ser Thr SerGly Ser Gly Lys Ser Ser Glu Gly Lys Gly (SEQ ID NO:11), Lys Glu Ser GlySer Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO:12),and Glu Ser Gly Ser Val Ser Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQID NO:13). Alternatively, a linker such as the (Gly-Gly-Gly-Gly-Ser)₃(SEQ ID NO:6) linker, although any sequence can be used, is mutagenizedor the amino acids in the linker are randomized, and using phage displayvectors or the methods of the invention, antibodies with differentlinkers are screened or selected for the highest affinity or most affecton phenotype. Examples of shorter linkers include fragments of the abovelinkers, and examples of longer linkers include combinations of thelinkers above, combinations of fragments of the linkers above, andcombinations of the linkers above with fragments of the linkers above.

[0091] Also preferred are immunoglobulin subunit polypeptides which arevariants or fragments of the above-described immunoglobulin subunitpolypeptides. Any variants or fragments which result in an antigenbinding fragment of an immunoglobulin molecule are contemplated. Suchvariants may be attached to the host cell surface, e.g., throughassociation with a naturally-occurring transmembrane domain, through areceptor-ligand interaction, or as a fusion with a heterologoustransmembrane domain, or may be secreted into the cell medium. Examplesof antigen binding fragments of immunoglobulin molecules are describedherein.

[0092] In those embodiments where the immunoglobulin subunit polypeptidecomprises a heavy chain polypeptide, any immunoglobulin heavy chain,from any animal species, is intended. Suitable andpreferredimmunoglobulin heavy chains are described herein. Immunoglobulin heavychains from vertebrates such as birds, especially chickens, fish, andmammals are included, with mammalian immunoglobulin heavy chains beingpreferred. Examples of mammalian immunoglobulin heavy chains includehuman, mouse, dog, cat, horse, goat, rat, sheep, cow, pig, guinea pig,camel, llama, and hamster immunoglobulin heavy chains. Of these, humanimmunoglobulin heavy chains are particularly preferred. Alsocontemplated are hybrid immunoglobulin heavy chains comprising portionsof heavy chains from one or more species, such as mouse/human hybridimmunoglobulin heavy chains, or “camelized” human immunoglobulin heavychains. Of the human immunoglobulin heavy chains, preferably, animmunoglobulin heavy chain of the present invention is selected from thegroup consisting of a μ heavy chain, i.e., the heavy chain of an IgMimmunoglobulin, a γ-1 heavy chain, i.e., the heavy chain of an IgG1immunoglobulin, a γ-2 heavy chain, i.e., the heavy chain of an IgG2immunoglobulin, a γ-3 heavy chain, i.e., the heavy chain of an IgG3immunoglobulin, a γ-4 heavy chain, i.e., the heavy chain of an IgG4immunoglobulin, an α-1 heavy chain, i.e., the heavy chain of an IgA1immunoglobulin, an α-2 heavy chain, i.e., the heavy chain of an IgA2immunoglobulin, and ε heavy chain, i.e., the heavy chain of animmunoglobulin, and a δ heavy chain, i.e., the heavy chain of an IgDimmunoglobulin. In certain embodiments, the preferred immunoglobulinheavy chains include membrane-bound forms of human μ, γ-1, γ-2, γ-3,γ-4, α-1, α-2, ε, and δ heavy chains. Especially preferred is a membranebound form of the human μ heavy chain.

[0093] Membrane bound forms of immunoglobulins are typically anchored tothe surface of cells by a transmembrane domain which is made part of theheavy chain polypeptide through alternative transcription terminationand splicing of the heavy chain messenger RNA. See, e.g., Roitt at page9.10. By “transmembrane domain” “membrane spanning region,” or relatedterms, which are used interchangeably herein, is meant the portion ofheavy chain polypeptide which is anchored into a cell membrane. Typicaltransmembrane domains comprise hydrophobic amino acids as discussed inmore detail below. By “intracellular domain,” “cytoplasmic domain,”“cytosolic region,” or related terms, which are used interchangeablyherein, is meant the portion of the polypeptide which is inside thecell, as opposed to those portions which are either anchored into thecell membrane or exposed on the surface of the cell. Membrane-boundforms of immunoglobulin heavy chain polypeptides typically comprise veryshort cytoplasmic domains of about three amino acids. A membrane-boundform of an immunoglobulin heavy chain polypeptide of the presentinvention preferably comprises the transmembrane and intracellulardomains normally associated with that immunoglobulin heavy chain, e.g.,the transmembrane and intracellular domains associated with μ and δheavy chains in pre-B cells, or the transmembrane and intracellulardomains associated with any of the immunoglobulin heavy chains inB-memory cells. However, it is also contemplated that heterologoustransmembrane and intracellular domains could be associated with a givenimmunoglobulin heavy chain polypeptide, for example, the transmembraneand intracellular domains of a μ heavy chain could be associated withthe extracellular portion of a γ heavy chain. Alternatively,transmembrane and/or cytoplasmic domains of an entirely heterologouspolypeptide could be used, for example, the transmembrane andcytoplasmic domains of a major histocompatibility molecule, a cellsurface receptor, a virus surface protein, chimeric domains, orsynthetic domains.

[0094] In those embodiments where the immunoglobulin subunit polypeptidecomprises a light chain polypeptide, any immunoglobulin light chain,from any animal species, is intended. Suitable and preferredimmunoglobulin light chains are described herein. Immunoglobulin lightchains from vertebrates such as birds, especially chickens, fish, andmammals are included, with mammalian immunoglobulin light chains beingpreferred. Examples of mammalian immunoglobulin light chains includehuman, mouse, dog, cat, horse, goat, rat, sheep, cow, pig, guinea pig,and hamster immunoglobulin light chains. Of these, human immunoglobulinlight chains are particularly preferred. Also contemplated are hybridimmunoglobulin light chains comprising portions of light chains from oneor more species, such as mouse/human hybrid immunoglobulin light chains.Preferred immunoglobulin light chains include human κ and λ lightchains. A pair of either light chain may associate with an identicalpair of any of the heavy chains to produce an immunoglobulin molecule,with the characteristic H₂L₂ structure which is well understood by thoseof ordinary skill in the art.

[0095] According to a preferred aspect of the invention, each member ofa library of polynucleotides as described herein, e.g., a first libraryof polynucleotides or a second library of polynucleotides, comprises (a)a first nucleic acid molecule encoding an immunoglobulin constant regioncommon to all members of the library, and (b) a second nucleic acidmolecule encoding an immunoglobulin variable region, where the secondnucleic acid molecule is directly upstream of and in-frame with thefirst nucleic acid molecule. Accordingly, an immunoglobulin subunitpolypeptide encoded by a member of a library of polynucleotides of thepresent invention, i.e., an immunoglobulin light chain or animmunoglobulin heavy chain encoded by such a polynucleotide, preferablycomprises an immunoglobulin constant region associated with animmunoglobulin variable region.

[0096] The constant region of a light chain encoded by the “firstnucleic acid molecule,” comprises about half of the subunit polypeptideand is situated C-terminal, i.e., in the latter half of the light chainpolypeptide. A light chain constant region, referred to herein as aC_(L) constant region, or, more specifically a C_(κ) constant region ora Cλ constant region, comprises about 110 amino acids held together in a“loop” by an interchain disulfide bond.

[0097] The constant region of a heavy chain encoded by the “firstnucleic acid molecule” comprises three quarters or more of the subunitpolypeptide, and is situated in the C-terminal, i.e., in the latterportion of the heavy chain polypeptide. The heavy chain constant region,referred herein as a C_(H) constant region, comprises either three orfour peptide loops or “domains” of about 110 amino acid each enclosed byinterchain disulfide bonds. More specifically, the heavy chain constantregions in human immunoglobulins include a Cμ constant region, a Cδconstant region, a Cγ constant region, a Cα constant region, and a Cεconstant region. Cγ, Cα, and Cδ heavy chains each contain three constantregion domains, referred to generally as C_(H)1, C_(H)2, and C_(H)3,while Cμ and Cε heavy chains contain four constant region domains,referred to generally as C_(H)1, C_(H)2, C_(H)3, and C_(H)4. Nucleicacid molecules encoding human immunoglobulin constant regions arereadily obtained from cDNA libraries derived from, for example, human Bcells or their precursors by methods such as PCR, which are well knownto those of ordinary skill in the art and further, are disclosed in theExamples, infra.

[0098] Immunoglobulin subunit polypeptides of the present invention eachcomprise an immunoglobulin variable region, encoded by the “secondnucleic acid molecule.” Within a library of polynucleotides, eachpolynucleotide will comprise the same constant region, but the librarywill contain a plurality, i.e., at least two, preferably at least 10,100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different variable regions. Asis well known by those of ordinary skill in the art, a light chainvariable region is encoded by rearranged nucleic acid molecules, eachcomprising a light chain V_(L) region, specifically a Vκ region or a Vλregion, and a light chain J region, specifically a Jκ region or a Jλregion. Similarly, a heavy chain variable region is encoded byrearranged nucleic acid molecules, each comprising a heavy chain V_(H)region, a D region and J region. These rearrangements take place at theDNA level upon cellular differentiation. Nucleic acid molecules encodingheavy and light chain variable regions may be derived, for example, byPCR from mature B cells and plasma cells which have terminallydifferentiated to express an antibody with specificity for a particularepitope. Furthermore, if antibodies to a specific antigen are desired,variable regions may be isolated from mature B cells and plasma cells ofan animal who has been immunized with that antigen, and has therebyproduced an expanded repertoire of antibody variable regions whichinteract with the antigen. Alternatively, if a more diverse library isdesired, variable regions may be isolated from precursor cells, e.g.,pre-B cells and immature B cells, which have undergone rearrangement ofthe immunoglobulin genes, but have not been exposed to antigen, eitherself or non-self. For example, variable regions might be isolated by PCRfrom normal human bone marrow pooled from multiple donors.Alternatively, variable regions may be synthetic, for example, made inthe laboratory through generation of synthetic oligonucleotides, or maybe derived through in vitro manipulations of germ line DNA resulting inrearrangements of the immunoglobulin genes.

[0099] In addition to first and second nucleic acid molecules encodingimmunoglobulin constant regions and variable regions, respectively, eachmember of a library of polynucleotides of the present invention asdescribed above may further comprise a third nucleic acid moleculeencoding a signal peptide directly upstream of and in frame with thesecond nucleic acid molecule encoding the variable region.

[0100] By “signal peptide” is meant a polypeptide sequence which, forexample, directs transport of nascent immunoglobulin polypeptide subunitto the surface of the host cells. Signal peptides are also referred toin the art as “signal sequences,” “leader sequences,” “secretory signalpeptides,” or “secretory signal sequences.” Signal peptides are normallyexpressed as part of a complete or “immature” polypeptide, and arenormally situated at the N-terminus. The common structure of signalpeptides from various proteins is commonly described as a positivelycharged n-region, followed by a hydrophobic h-region and a neutral butpolar c-region. In many instances the amino acids comprising the signalpeptide are cleaved off the protein once its final destination has beenreached, to produce a “mature” form of the polypeptide. The cleavage iscatalyzed by enzymes known as signal peptidases. The (−3,−1)-rule statesthat the residues at positions −3 and −1 (relative to the cleavage site)must be small and neutral for cleavage to occur correctly. See, e.g.,McGeoch, Virus Res. 3:271-286 (1985), and von Heinje, Nucleic Acids Res.14:4683-4690 (1986).

[0101] All cells, including host cells of the present invention, possessa constitutive secretory pathway, where proteins, including secretedimmunoglobulin subunit polypeptides destined for export, are secretedfrom the cell. These proteins pass through the ER-Golgi processingpathway where modifications may occur. If no further signals aredetected on the protein it is directed to the cells surface forsecretion. Alternatively, immunoglobulin subunit polypeptides can end upas integral membrane components expressed on the surface of the hostcells. Membrane-bound forms of immunoglobulin subunit polypeptidesinitially follow the same pathway as the secreted forms, passing throughto the ER lumen, except that they are retained in the ER membrane by thepresence of stop-transfer signals, or “transmembrane domains.”Transmembrane domains are hydrophobic stretches of about 20 amino acidresidues that adopt an alpha-helical conformation as they transverse themembrane. Membrane embedded proteins are anchored in the phospholipidbilayer of the plasma membrane. As with secreted proteins, theN-terminal region of transmembrane proteins have a signal peptide thatpasses through the membrane and is cleaved upon exiting into the lumenof the ER. Transmembrane forms of immunoglobulin heavy chainpolypeptides utilize the same signal peptide as the secreted forms.

[0102] A signal peptide of the present invention may be either anaturally-occurring immunoglobulin signal peptide, i.e., encoded by asequence which is part of a naturally occurring heavy or light chaintranscript, or a functional derivative of that sequence that retains theability to direct the secretion of the immunoglobulin subunitpolypeptide that is operably associated with it. Alternatively, aheterologous signal peptide, or a functional derivative thereof, may beused. For example, a naturally-occurring immunoglobulin subunitpolypeptide signal peptide may be substituted with the signal peptide ofhuman tissue plasminogen activator or mouse β-glucuronidase.

[0103] Signal sequences, transmembrane domains, and cytosolic domainsare known for a wide variety of membrane bound proteins. These sequencesmay be used accordingly, either together as pairs (e.g., signal sequenceand transmembrane domain, or signal sequence and cytosolic domain, ortransmembrane domain and cytosolic domain) or threesomes from aparticular protein, or with each component being taken from a differentprotein, or alternatively, the sequences may be synthetic, and derivedentirely from consensus as artificial delivery domains, as mentionedabove.

[0104] Particularly preferred signal sequences and transmembrane domainsinclude, but are not limited to, those derived from CD8, ICAM-2, IL-8R,CD4 and LFA-1. Additional useful sequences include sequences from: 1)class I integral membrane proteins such as IL-2 receptor beta-chain(residues 1-26 are the signal sequence, 241-265 are the transmembraneresidues; see Hatakeyama et al, Science 244:551 (1989) and von Heijne etal, Eur. J. Biochem. 174:671 (1988)) and insulin receptor beta-chain(residues 1-27 are the signal, 957-959, are the transmembrane domain and960-1382 are the cytoplasmic domain; see Hatakeyama supra, and Ebina etal., Cell 40:747 (1985)); 2) class II integral membrane proteins such asneutral endopeptidase (residues 29-51 are the transmembrane domain, 2-28are the cytoplasmic domain; see Malfroy et al., Biochem. Biophys. Res.Commun. 144:59 (1987)); 3) type III proteins such as human cytochromeP450 NF25 (Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). In this alternative, CD8 and ICAM-2are particularly preferred. For example, the signal sequences from CD8and ICAM-2 lie at the extreme 5′ end of the transcript. These consist ofthe amino acids 1-32 in the case of CD8 (Nakauchi et al., PNAS USA82:5126 (1985)) and 1-21 in the case of ICAM-2 (Staunton et al., Nature(London) 339:61 (1989)). These transmembrane domains are encompassed byamino acids 145-195 from CD8 (Nakauchi, supra) and 224-256 from ICAM-2(Staunton, supra).

[0105] Alternatively, membrane anchoring domains include the GPI anchor,which results in a covalent bond between the molecule and the lipidbilayer via a glycosyl-phosphatidylinositol bond for example in DAF (seeHomans et al., Nature 333(6170):269-72 (1988), and Moran et al., J.Biol. Chem. 266:1250 (1991)). In order to do this, the GPI sequence fromThy-1 can be cassetted 3′ of the immunoglobulin or immunoglobulinfragment in place of a transmembrane sequence.

[0106] Similarly, myristylation sequences can serve as membraneanchoring domains. It is known that the myristylation of c-src recruitsit to the plasma membrane. This is a simple and effective method ofmembrane localization, given that the first 14 amino acids of theprotein are solely responsible for this function (see Cross et al., Mol.Cell. Biol. 4(9) 1834 (1984); Spencer et al., Science 262:1019 1024(1993)). This motif has already been shown to be effective in thelocalization of reporter genes and can be used to anchor the zeta chainof the TCR. This motif is placed 5′ of the immunoglobulin orimmunoglobulin fragment in order to localize the construct to the plasmamembrane. Other modifications such as palmitoylation can be used toanchor constructs in the plasma membrane; for example, palmitoylationsequences from the G protein-coupled receptor kinase GRK6 sequence(Stoffel et al, J. Biol. Chem 269:27791 (1994)); from rhodopsin(Barnstable et al., J. Mol. Neurosci. 5(3):207 (1994)); and the p21H-ras 1 protein (Capon et al., Nature 302:33 (1983)).

[0107] In addition to first and second nucleic acid molecules encodingimmunoglobulin constant regions and variable regions, respectively, eachmember of a library of polynucleotides of the present invention asdescribed above may further comprise additional nucleic acid moleculeencoding heterologous polypeptides. Such additional polynucleotides maybe in addition to or as an alternative of the third nucleic acidmolecule encoding a signal peptide. Such additional nucleic acidmolecules encoding heterologous polypeptides may be upstream of ordownstream from the nucleic acid molecules encoding the variable chainregion or the heavy chain region.

[0108] A heterologous polypeptide encoded by an additional nucleic acidmolecule may be a rescue sequence. A rescue sequence is a sequence whichmay be used to purify or isolate either the immunoglobulin or fragmentthereof or the polynucleotide encoding it. Thus, for example, peptiderescue sequences include purification sequences such as the 6-His tagfor use with Ni affinity columns and epitope tags for detection,immunoprecipitation, orFACS (fluorescence-activated cell sorting).Suitable epitope tags include myc (for use with commercially available9E10 antibody), the BSP biotinylation target sequence of the bacterialenzyme BirA, flu tags, LacZ, and GST. The additional nucleic acidmolecule may also encode a peptide linker.

[0109] In a preferred embodiment, combinations of heterologouspolypeptides are used. Thus, for example, any number of combinations ofsignal sequences, rescue sequences, and stability sequences may be used,with or without linker sequences. One can cassette in various fusionpolynucleotides encoding heterologous polypeptides 5′ and 3 of theimmunoglobulin or fragment thereof-encoding polynucleotide. As will beappreciated by those in the art, these modules of sequences can be usedin a large number of combinations and variations.

[0110] The polynucleotides comprised in the first and second librariesare introduced into suitable host cells. Suitable host cells arecharacterized by being capable of expressing immunoglobulin moleculesattached to their surface. Polynucleotides may be introduced into hostcells by methods which are well known to those of ordinary skill in theart. Suitable and preferred introduction methods are disclosed herein.

[0111] As is easily appreciated, introduction methods vary depending onthe nature of the vector in which the polynucleotide libraries areconstructed. For example, DNA plasmid vectors may be introduced intohost cells, for example, by lipofection (such as with anionic liposomes(see, e.g., Felgner et al., 1987 Proc. Natl. Acad Sci. U.S.A. 84:7413 orcationic liposomes (see, e.g., Brigham, K. L. et al. Am. J Med Sci.298(4):278-2821(1989); U.S. Pat. No. 4,897,355 (Eppstein, et al.)), byelectroporation, by calcium phosphate precipitation (see generally,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989), by protoplastfusion, by spheroplast fusion, or by the DEAE dextran method (Sussman etal., Cell. Biol. 4:1641-1643 (1984)). The above references areincorporated herein by reference in their entireties.

[0112] When the selected method is lipofection, the nucleic acid can becomplexed with a cationic liposome, such as DOTMA:DOPE, DOTMA, DOPE,DC-cholesterol, DOTAP, Transfectam® (Promega), Tfx® (Promega), LipoTAXI™(Stratagene), PerFect Lipid™ (Invitrogen), SuperFect™ (Qiagen). When thenucleic acid is transfected via an anionic liposome, the anionicliposome can encapsulate the nucleic acid. Preferably, DNA is introducedby liposome-mediated transfection using the manufacturer's protocol(such as for Lipofectamine; Life Technologies Incorporated).

[0113] Where the plasmid is a virus vector, introduction into host cellsis most conveniently carried out by standard infection. However, in manycases viral nucleic acids may be introduced into cells by any of themethods described above, and the viral nucleic acid is “infectious,”i.e., introduction of the viral nucleic acid into the cell, withoutmore, is sufficient to allow the cell to produce viable progeny virusparticles. It is noted, however, that certain virus nucleic acids, forexample, poxvirus nucleic acids, are not infectious, and therefore mustbe introduced with additional elements provided, for example, by a virusparticle enclosing the viral nucleic acid, by a cell which has beenengineered to produce required viral elements, or by a helper virus.

[0114] The first and second libraries of polynucleotides may beintroduced into host cells in any order, or simultaneously. For example,if both the first and second libraries of polynucleotides areconstructed in virus vectors, whether infectious or inactivated, thevectors maybe introduced by simultaneous infection as a mixture, or maybe introduced in consecutive infections. If one library is constructedin a virus vector, and the other is constructed in a plasmid vector,introduction might be carried out most conveniently by introduction ofone library before the other.

[0115] Following introduction into the host cells of the first andsecond libraries of polynucleotides, expression of immunoglobulinmolecules, or antigen-specific fragments thereof, is permitted to occureither on the membrane surface of said host cells, or through secretioninto the cell medium. By “permitting expression” is meant allowing thevectors which have been introduced into the host cells to undergotranscription and translation of the immunoglobulin subunitpolypeptides, preferably allowing the host cells to transport fullyassembled immunoglobulin molecules, or antigen-specific fragmentsthereof, to the membrane surface or into the cell medium. Typically,permitting expression requires incubating the host cells into which thepolynucleotides have been introduced under suitable conditions to allowexpression. Those conditions, and the time required to allow expressionwill vary based on the choice of host cell and the choice of vectors, asis well known by those of ordinary skill in the art.

[0116] In certain embodiments, host cells which have been allowed toexpress immunoglobulin molecules on their surface, or solubleimmunoglobulin molecules secreted into the cell medium are thencontacted with an antigen. As used herein, an “antigen” is any moleculethat can specifically bind to an antibody, immunoglobulin molecule, orantigen-specific fragment thereof. By “specifically bind” is meant thatthe antigen binds to the CDR of the antibody. The portion of the antigenwhich specifically interacts with the CDR is an “epitope,” or an“antigenic determinant.” An antigen may comprise a single epitope, buttypically, an antigen comprises at least two epitopes, and can includeany number of epitopes, depending on the size, conformation, and type ofantigen.

[0117] Antigens are typically peptides or polypeptides, but can be anymolecule or compound. For example, an organic compound, e.g.,dinitrophenol or DNP, a nucleic acid, a carbohydrate, or a mixture ofany of these compounds either with or without a peptide or polypeptidecan be a suitable antigen. The minimum size of a peptide or polypeptideepitope is thought to be about four to five amino acids. Peptide orpolypeptide epitopes preferably contain at least seven, more preferablyat least nine and most preferably between at least about 15 to about 30amino acids. Since a CDR can recognize an antigenic peptide orpolypeptide in its tertiary form, the amino acids comprising an epitopeneed not be contiguous, and in some cases, may not even be on the samepeptide chain. In the present invention, peptide or polypeptide antigenspreferably contain a sequence of at least 4, at least 5, at least 6, atleast 7, more preferably at least 8, at least 9, at least 10, at least15, at least 20, at least 25, and, most preferably, between about 15 toabout 30 amino acids. Preferred peptides or polypeptides comprising, oralternatively consisting of, antigenic epitopes are at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 aminoacid residues in length. The antigen may be in any form and may be free,for example dissolved in a solution, or may be attached to anysubstrate. Suitable and preferred substrates are disclosed herein. Incertain embodiments, an antigen may be part of an antigen-expressingpresenting cell as described in more detail below.

[0118] It is to be understood that immunoglobulin molecules specific forany antigen may be produced according to the methods of the presentinvention. Preferred antigens are “self” antigens, i.e., antigensderived from the same species as the immunoglobulin molecules produced.As an example, it might be desired to produce human antibodies directedto human tumor antigens such as, but not limited to, a CEA antigen, aGM2 antigen, a Tn antigen, an sTn antigen, a Thompson-Friedenreichantigen (TF), a Globo H antigen, an Le(y) antigen, a MUC1 antigen, aMUC2 antigen, a MUC3 antigen, a MUC4 antigen, a MUC5AC antigen, a MUC5Bantigen, a MUC7 antigen, a carcinoembryonic antigen, a beta chain ofhuman chorionic gonadotropin (hCG beta) antigen, a HER2/neu antigen, aPSMA antigen, a EGFRvIII antigen, a KSA antigen, a PSA antigen, a PSCAantigen, a GP100 antigen, a MAGE 1 antigen, a MAGE 2 antigen, a TRP 1antigen, a TRP 2 antigen, and a tyrosinase antigen. Other desired “self”antigens include, but are not limited to, cytokines, receptors, ligands,glycoproteins, and hormones.

[0119] It is also contemplated to produce antibodies directed toantigens on infectious agents. Examples of such antigens include, butare not limited to, bacterial antigens, viral antigens, parasiteantigens, and fungal antigens. Examples of viral antigens include, butare not limited to, adenovirus antigens, alphavirus antigens,calicivirus antigens, e.g., a calicivirus capsid antigen, coronavirusantigens, distemper virus antigens, Ebola virus antigens, enterovirusantigens, flavivirus antigens, hepatitis virus (A-E) antigens, e.g., ahepatitis B core or surface antigen, herpesvirus antigens, e.g., aherpes simplex virus or varicella zoster virus glycoprotein antigen,immunodeficiency virus antigens, e.g., a human immunodeficiency virusenvelope or protease antigen, infectious peritonitis virus antigens,influenza virus antigens, e.g., an influenza A hemagglutinin orneuraminidase antigen, leukemia virus antigens, Marburg virus antigens,oncogenic virus antigens, orthomyxovirus antigens, papilloma virusantigens, parainfluenza virus antigens, e.g.,hemagglutinin/neuraminidase antigens, paramyxovirus antigens, parvovirusantigens, pestivirus antigens, picorna virus antigens, e.g., apoliovirus capsid antigen, rabies virus antigens, e.g., a rabies virusglycoprotein G antigen, reovirus antigens, retrovirus antigens,rotavirus antigens, as well as other cancer-causing or cancer-relatedvirus antigens.

[0120] Examples of bacterial antigens include, but are not limited to,Actinomyces, antigens Bacillus antigens, Bacteroides antigens,Bordetella antigens, Bartonella antigens, Borrelia antigens, e.g., a B.bergdorferi OspA antigen, Brucella antigens, Campylobacter antigens,Capnocytophaga antigens, Chlamydia antigens, Clostridium antigens,Corynebacterium antigens, Coxiella antigens, Dennatophilus antigens,Enterococcus antigens, Ehrlichia antigens, Escherichia antigens,Francisella antigens, Fusobacterium antigens, Haemobartonella antigens,Haemophilus antigens, e.g., H. influenzae typeb outer membrane proteinantigens, Helicobacter antigens, Klebsiella antigens, L-form bacteriaantigens, Leptospira antigens, Listeria antigens, Mycobacteria antigens,Mycoplasma antigens, Neisseria antigens, Neorickettsia antigens,Nocardia antigens, Pasteurella antigens, Peptococcus antigens,Peptostreptococcus antigens, Pneumococcus antigens, Proteus antigens,Pseudomonas antigens, Rickettsia antigens, Rochalimaea antigens,Salmonella antigens, Shigella antigens, Staphylococcus antigens,Streptococcus antigens, e.g., S. pyogenes M protein antigens, Treponemaantigens, and Yersinia antigens, e.g., YpestisF1 and V antigens.

[0121] Examples of fungal antigens include, but are not limited to,Absidia antigens, Acremonium antigens, Alternaria antigens, Aspergillusantigens, Basidiobolus antigens, Bipolaris antigens, Blastomycesantigens, Candida antigens, Coccidioides antigens, Conidiobolusantigens, Cryptococcus antigens, Curvalaria antigens, Epidermophytonantigens, Exophiala antigens, Geotrichum antigens, Histoplasma antigens,Madurella antigens, Malassezia antigens, Microsporum antigens,Moniliella antigens, Mortierella antigens, Mucor antigens, Paecilomycesantigens, Penicillium antigens, Phialemonium antigens, Phialophoraantigens, Prototheca antigens, Pseudallescheria antigens,Pseudomicrodochium antigens, Pythium antigens, Rhinosporidium antigens,Rhizopus antigens, Scolecobasidium antigens, Sporothrix antigens,Stemphylium antigens, Trichophyton antigens, Trichosporon antigens, andXylohypha antigens.

[0122] Examples of protozoan parasite antigens include, but are notlimited to, Babesia antigens, Balantidium antigens, Besnoitia antigens,Cryptosporidium antigens, Eimeri antigens a antigens, Encephalitozoonantigens, Entamoeba antigens, Giardia antigens, Hammondia antigens,Hepatozoon antigens, Isospora antigens, Leishmania antigens,Microsporidia antigens, Neospora antigens, Nosema antigens,Pentatrichomonas antigens, Plasmodium antigens, e.g., P. falciparumcircumsporozoite (PfCSP), sporozoite surface protein 2 (PfSSP2),carboxyl terminus of liver state antigen 1 (PFLSA-1 c-term), andexported protein 1 (PfExp-1) antigens, Pneumocystis antigens,Sarcocystis antigens, Schistosoma antigens, Theileria antigens,Toxoplasma antigens, and Trypanosoma antigens. Examples of helminthparasite antigens include, but are not limited to, Acanthocheilonemaantigens, Aelurostrongylus antigens, Ancylostoma antigens,Angiostrongylus antigens, Ascaris antigens, Brugia antigens, Bunostomumantigens, Capillaria antigens, Chabertia antigens, Cooperia antigens,Crenosoma antigens, Dictyocaulus antigens, Dioctophyme antigens,Dipetalonema antigens, Diphyllobothrium antigens, Diplydium antigens,Dirofilaria antigens, Dracunculus antigens, Enterobius antigens,Filaroides,antigens Haemonchus antigens, Lagochilascaris antigens, Loaantigens, Mansonella antigens, Muellerius antigens, Nanophyetusantigens, Necator antigens, Nematodirus antigens, Oesophagostomumantigens, Onchocerca antigens, Opisthorchis antigens, Ostertagiaantigens, Parafilaria antigens, Paragonimus antigens, Parascarisantigens, Physaloptera antigens, Protostrongylus antigens, Setariaantigens, Spirocerca,antigens Spirometra antigens, Stephanofilariaantigens, Strongyloides antigens, Strongylus antigens, Thelaziaantigens, Toxascaris antigens, Toxocara antigens, Trichinella antigens,Trichostrongylus antigens, Trichuris antigens. Uncinaria antigens, andWuchereria antigens.

[0123] In certain selection and screening schemes in whichimmunoglobulin molecules are expressed on the surface of host cells, thehost cells of the present invention are “contacted” with antigen by amethod which will allow an antigen, which specifically recognizes a CDRof an immunoglobulin molecule expressed on the surface of the host cell,to bind to the CDR, thereby allowing the host cells which specificallybind the antigen to be distinguished from those host cells which do notbind the antigen. Any method which allows host cells expressing anantigen-specific antibody to interact with the antigen is included. Forexample, if the host cells are in suspension, and the antigen isattached to a solid substrate, cells which specifically bind to theantigen will be trapped on the solid substrate, allowing those cellswhich do not bind the antigen to be washed away, and the bound cells tobe subsequently recovered. Alternatively, if the host cells are attachedto a solid substrate, and by specifically binding antigen cells arecaused to be released from the substrate (e.g., by cell death), they canbe recovered from the cell supernatant. Preferred methods by which toallow host cells of the invention to contact antigen, especially usinglibraries constructed in vaccinia virus vectors by trimolecularrecombination, are disclosed herein.

[0124] In a preferred screening method for the detection ofantigen-specific immunoglobulin molecules expressed on the surface ofhost cells, the host cells of the present invention are incubated with aselecting antigen that has been labeled directly withfluorescein-5-isothiocyanate (FITC) or indirectly with biotin thendetected with FITC-labeled streptavidin. Other fluorescent probes can beemployed which will be familiar to those practiced in the art. Duringthe incubation period, the labeled selecting antigen binds theantigen-specific immunoglobulin molecules. Cells expressing an antibodyreceptor for a specific fluorescence tagged antigen can be selected byfluorescence activated cell sorting, thereby permitting the host cellswhich specifically bind the antigen to be distinguished from those hostcells which do not bind the antigen. With the advent of cell sorterscapable of sorting more than 1×10⁸ cells per hour, it is feasible toscreen large numbers of cells infected with recombinant vaccinialibraries of immunoglobulin genes to select the subset of cells thatexpress specific antibody receptors to the selecting antigen.

[0125] After recovery of host cells which specifically bind antigen,polynucleotides of the first library are recovered from those hostcells. By “recovery” is meant a crude separation of a desired componentfrom those components which are not desired. For example, host cellswhich bind antigen are “recovered” based on their detachment from asolid substrate, and polynucleotides of the first library are recoveredfrom those cells by crude separation from other cellular components. Itis to be noted that the term “recovery” does not imply any sort ofpurification or isolation away from viral and other components. Recoveryof polynucleotides may be accomplished by any standard method known tothose of ordinary skill in the art. In a preferred aspect, thepolynucleotides are recovered by harvesting infectious virus particles,for example, particles of a vaccinia virus vector into which the firstlibrary has been constructed, which were contained in those host cellswhich bound antigen.

[0126] In certain screening schemes in which immunoglobulin moleculesare fully secreted from the surface of host cells, the cell medium inwhich pools of host cells are cultured, i.e., “conditioned medium,” maybe “contacted” with antigen by a method which will allow an antigenwhich specifically recognizes a CDR of an immunoglobulin molecule tobind to the CDR, and which further allows detection of theantigen-antibody interaction. Such methods include, but are not limitedto, immunoblots, ELISA assays, RIA assays, RAST assays, andimmunofluorescence assays. Alternatively, the conditioned medium issubjected to a functional assay for specific antibodies. Examples ofsuch assays include, but are not limited to, virus neutralization assays(for antibodies directed to specific viruses), bacterialopsonization/phagocytosis assays (for antibodies directed to specificbacteria), antibody-dependent cellular cytotoxicity (ADCC) assays,assays to detect inhibition or facilitation of certain cellularfunctions, assays to detect IgE-mediated histamine release from mastcells, hemagglutination assays, and hemagglutination inhibition assays.Such assays will allow detection of antigen-specific antibodies withdesired functional characteristics.

[0127] After the identification of conditioned medium pools containingimmunoglobulin molecules which specifically bind antigen, or which havedesired functional characteristics, further screening steps are carriedout until host cells which produce the desired immunoglobulin moleculesare recovered, and then polynucleotides of the first library arerecovered from those host cells.

[0128] As will be readily appreciated by those of ordinary skill in theart, identification of polynucleotides encoding immunoglobulinsubunitpolypeptides may require two or more rounds of selection asdescribed above, and will necessarily require two or more rounds ofscreening as described above. A single round of selection may notnecessarily result in isolation of a pure set of polynucleotidesencoding the desired first immunoglobulin subunit polypeptides; themixture obtained after a first round may be enriched for the desiredpolynucleotides but may also be contaminated with non-target insertsequences. Screening assays described herein identify pools containingthe reactive host cells, and/or immunoglobulin molecules, but such poolswill also contain non-reactive species. Therefore, the reactive poolsare further fractionated and subjected to further rounds of screening.Thus, identification of polynucleotides encoding a first immunoglobulinsubunit polypeptide which, in association with a second immunoglobulinsubunit polypeptide, is capable of forming a desired immunglobulinmolecule, or antigen-specific fragment thereof, may require or benefitfrom several rounds of selection and/or screening, which thus increasesthe proportion of cells containing the desired polynucleotides.Accordingly, this embodiment further provides that the polynucleotidesrecovered after the first round be introduced into a second populationof cells and be subjected to a second round of selection.

[0129] Accordingly, the first selection step, as described, may, or mustbe repeated one or more times, thereby enriching for the polynucleotidesencoding the desired immunoglobulin subunit polypeptides. In order torepeat the first step of this embodiment, those polynucleotides, orpools of polynucleotides, recovered as described above are introducedinto a population of host cells capable of expressing the immunoglobulinmolecules encoded by the polynucleotides in the library. The host cellsmay be of the same type used in the first round of selection, or may bea different host cell, as long as they are capable of expressing theimmunoglobulin molecules. The second library of polynucleotides are alsointroduced into these host cells, and expression of immunoglobulinmolecules, or antigen-specific fragments thereof, on the membranesurface of said host cells, or in the cell medium, is permitted. Thecells or condition medium are similarly contacted with antigen, or themedium is tested in a functional assay, and polynucleotides of the firstlibrary are again recovered from those cells or pools of host cellswhich express an immunoglobulin molecule that specifically bindsantigen, and/or has a desired functional characteristic. These steps maybe repeated one or more times, resulting in enrichment forpolynucleotides derived from the first library which encode animmunoglobulin subunit polypeptide which, as part of an immunoglobulinmolecule, or antigen-specific fragment thereof, specifically binds theantigen and/or has a desired functional characteristic.

[0130] Following suitable enrichment for the desired polynucleotidesfrom the first library as described above, those polynucleotides whichhave been recovered are “isolated,” i.e., they are substantially removedfrom their native environment and are largely separated frompolynucleotides in the library which do not encode antigen-specificimmunoglobulin subunit polypeptides. For example, cloned polynucleotidescontained in a vector are considered isolated for the purposes of thepresent invention. It is understood that two or more differentimmunoglobulin subunit polypeptides which specifically bind the sameantigen can be recovered by the methods described herein. Accordingly, amixture of polynucleotides which encode polypeptides binding to the sameantigen is also considered to be “isolated. ” Further examples ofisolated polynucleotides include those maintained in heterologous hostcells or purified (partially or substantially) DNA molecules insolution. However, a polynucleotide contained in a clone that is amember of a mixed library and that has not been isolated from otherclones of the library, e.g., by virtue of encoding an antigen-specificimmunoglobulin subunit polypeptide, is not “isolated” for the purposesof this invention. For example, a polynucleotide contained in a virusvector is “isolated” after it has been recovered, and plaque purified,and a polynucleotide contained in a plasmid vector is isolated after ithas been expanded from a single bacterial colony.

[0131] Given that an antigen may comprise two or more epitopes, andseveral different immunoglobulin molecules may bind to any givenepitope, it is contemplated that several suitable polynucleotides, e.g.,two, three, four, five, ten, 100 or more polynucleotides, may berecovered from the first step of this embodiment, all of which mayencode an immunoglobulin subunit polypeptide which, when combined with asuitable immunoglobulin subunit polypeptide encoded by a polynucleotideof the second library, will form an immunoglobulin molecule, or antigenbinding fragment thereof, capable of specifically binding the antigen ofinterest. It is contemplated that each different polynucleotiderecovered from the first library would be separately isolated. However,these polynucleotides may be isolated as a group of polynucleotideswhich encode polypeptides with the same antigen specificity, and thesepolynucleotides may be “isolated” together. Such mixtures ofpolynucleotides, whether separately isolated or collectively isolated,may be introduced into host cells in the second step, as explainedbelow, either individually, or with two, three, four, five, ten, 100 ormore of the polynucleotides pooled together.

[0132] Once one or more suitable polynucleotides from the first libraryare isolated, in the second step of this embodiment, one or morepolynucleotides are identified in the second library which encodeimmunoglobulin subunit polypeptides which are capable of associatingwith the immunoglobulin subunit polypeptide(s) encoded by thepolynucleotides isolated from the first library to form animmunoglobulin molecule, or antigen-binding fragment thereof, whichspecifically binds an antigen of interest, or has a desired functionalcharacteristic.

[0133] Accordingly, the second step comprises introducing into apopulation of host cells capable of expressing an immunoglobulinmolecule the second library of polynucleotides encoding a secondimmunoglobulin subunit polypeptide, introducing into the same populationof host cells at least one of the polynucleotides isolated from thefirst library as described above, permitting expression ofimmunoglobulin molecules, or antigen-specific fragments thereof, on thesurface of the host cells, or fully secreted into the cell medium,contacting those host cells, or conditioned medium in which the hostcells were grown, with the specific antigen of interest, or subjectingthe conditioned medium to a functional assay, and recoveringpolynucleotides of the second library from those host cells which bindthe antigen of interest, or those host cells which were grown in theconditioned medium which exhibits a desired reactivity. The second stepis thus carried out very similarly to the first step, except that thesecond immunoglobulin subunit polypeptides encoded by thepolynucleotides of the second library are combined in the host cellswith just those polynucleotides isolated from the first library. Asmentioned above, a single cloned polynucleotide isolated from the firstlibrary may be used, or alternatively a pool of several polynucleotidesisolated from the first library may be introduced simultaneously. Aswith the first step described above, one or more rounds of enrichmentare carried out, i.e., either selection or screening of successivelysmaller pools, thereby enriching for polynucleotides of the secondlibrary which encode a second immunoglobulin subunit polypeptide which,as part of an immunoglobulin molecule, or antigen-specific fragmentthereof, specifically binds the antigen of interest, or exhibits adesired functional characteristic. Also as with the first step, one ormore desired polynucleotides from the second library are then isolated.If a pool of isolated polynucleotides is used in the earlier rounds ofenrichment during the second step, preferred subsequent enrichment stepsmay utilize smaller pools of polynucleotides isolated from the firstlibrary, or even more preferably individual cloned polynucleotidesisolated from the first library. For any individual polynucleotideisolated from the first library which is then used in the selectionprocess for polynucleotides of the second library, it is possible thatseveral, i.e. two, three, four, five, ten, 100, or more polynucleotidesmay be isolated from the second library which encode a secondimmunoglobulin subunit polypeptide capable of associating with a firstimmunoglobulin subunit polypeptide encoded by a polynucleotide isolatedfrom the first library to form an immunoglobulin molecule, or antigenbinding fragment thereof, which specifically binds the antigen ofinterest, or exhibits a desired functional characteristic.

[0134] The selection/screening methods for libraries encodingsingle-chain fragments require only one library rather than first andsecond libraries, and only one selection/screening step is necessary.Similar to each of the two-steps for the immunoglobulins this one-stepselection/screening method may also benefit from two or more rounds ofenrichment.

[0135] Vectors. In constructing antibody libraries in eukaryotic cells,any standard vector which allows expression in eukaryotic cells may beused. For example, the library could be constructed in a virus, plasmid,phage, or phagemid vector as long as the particular vector chosencomprises transcription and translation regulatory regions capable offunctioning in eukaryotic cells. However, antibody libraries asdescribed above are preferably constructed in eukaryotic virus vectors.

[0136] Eukaryotic virus vectors may be of any type, e.g., animal virusvectors or plant virus vectors. The naturally-occurring genome of thevirus vector may be RNA, either positive strand, negative strand, ordouble stranded, or DNA, and the naturally-occurring genomes may beeither circular or linear. Of the animal virus vectors, those thatinfect either invertebrates, e.g., insects, protozoans, or helminthparasites; or vertebrates, e.g., mammals, birds, fish, reptiles, andamphibians are included. The choice of virus vector is limited only bythe maximum insert size, and the level of protein expression achieved.Suitable virus vectors are those that infect yeast and other fungalcells, insect cells, protozoan cells, plant cells, bird cells, fishcells, reptilian cells, amphibian cells, or mammalian cells, withmammalian virus vectors being particularly preferred. Any standard virusvector could be used in the present invention, including, but notlimited to poxvirus vectors (e.g., vaccinia virus), herpesvirus vectors(e.g., herpes simplex virus), adenovirus vectors, baculovirus vectors,retrovirus vectors, picorna virus vectors (e.g., poliovirus), alphavirusvectors (e.g., sindbis virus), and enterovirus vectors (e.g.,mengovirus). DNA virus vectors, e.g., poxvirus, herpes virus,baculovirus, and adenovirus are preferred. As described in more detailbelow, the poxviruses, particularly orthopoxviruses, and especiallyvaccinia virus, are particularly preferred. In a preferred embodiment,host cells are utilized which are permissive for the production ofinfectious viral particles of whichever virus vector is chosen. Manystandard virus vectors, such as vaccinia virus, have a very broad hostrange, thereby allowing the use of a large variety of host cells.

[0137] As mentioned supra, the first and second libraries of theinvention may be constructed in the same vector, or may be constructedin different vectors. However, in preferred embodiments, the first andsecond libraries are prepared such that polynucleotides of the firstlibrary can be conveniently recovered, e.g., separated, from thepolynucleotides of the second library in the first step, and thepolynucleotides of the second library can be conveniently recovered fromthe polynucleotides of the first library in the second step. Forexample, in the first step, if the first library is constructed in avirus vector, and the second library is constructed in a plasmid vector,the polynucleotides of the first library are easily recovered asinfectious virus particles, while the polynucleotides of the secondlibrary are left behind with cellular debris. Similarly, in the secondstep, if the second library is constructed in a virus vector, while thepolynucleotides of the first library isolated in the first step areintroduced in a plasmid vector, infectious virus particles containingpolynucleotides of the second library are easily recovered.

[0138] When the second library of polynucleotides, or thepolynucleotides isolated from the first library are introduced into hostcells in a plasmid vector, it is preferred that the immunoglobulinsubunit polypeptides encoded by polynucleotides comprised in suchplasmid vectors be operably associated with transcriptional regulatoryregions which are driven by proteins encoded by virus vector whichcontains the other library. For example, if the first library isconstructed in a poxvirus vector, and the second library is constructedin a plasmid vector, it is preferred that the polynucleotides encodingthe second immunoglobulin subunit polypeptides constructed in theplasmid library be operably associated with a transcriptional controlregion, preferably a promoter, which functions in the cytoplasm ofpoxvirus-infected cells. Similarly in the second step, if it is desiredto insert the polynucleotides isolated from the first library into aplasmid vector, and the second library is constructed in a poxvirusvector, it is preferred that polynucleotides isolated from the firstlibrary and inserted into plasmids be operably associated with atranscriptional regulatory region, preferably a promoter, whichfunctions in the cytoplasm of poxvirus-infected cells. Suitable andpreferred examples of such transcriptional control regions are disclosedherein. In this way, the polynucleotides of the second library are onlyexpressed in those cells which have also been infected by a poxvirus.

[0139] However, it is convenient to be able to maintain both the firstand second libraries, as well as those polynucleotides isolated from thefirst library, in just a virus vector rather than having to maintain oneor both of the libraries in two different vector systems. Accordingly,the present invention provides that samples of the first or secondlibraries, maintained in a virus vector, are inactivated such that thevirus vector infects cells and the genome of virus vector istranscribed, but the vector is not replicated, i.e., when the virusvector is introduced into cells, gene products carried on the virusgenome, e.g., immunoglobulin subunit polypeptides, are expressed, butinfectious virus particles are not produced.

[0140] In a preferred aspect, inactivation of either the first or secondlibrary constructed in a eukaryotic virus vector is carried out bytreating a sample of the library constructed in a virus vector with4′-aminomethyl-trioxsalen (psoralen) and then exposing the virus vectorto ultraviolet (UV) light. Psoralen and UV inactivation of viruses iswell known to those of ordinary skill in the art. See, e.g., Tsung, K.,et al., J. Virol. 70:165-171 (1996), which is incorporated herein byreference in its entirety.

[0141] Psoralen treatment typically comprises incubating a cell-freesample of the virus vector with a concentration of psoralen ranging fromabout 0.1 μg/ml to about 20 μg/ml, preferably about 1 μg/ml to about17.5 μg/ml, about 2.5 μg/ml to about 15 μg/ml, about 5 μg/ml to about12.5 μg/ml, about 7.5 μg/ml to about 12.5 μg/ml, or about 9 μg/ml toabout 11 μg/ml. Accordingly, the concentration of psoralen may be about0.1 μg/ml, 0.5 μg/ml, 1 μg/ml, 2 μg/ml, 3 μg/ml, 4 μg/ml, 5 μg/ml, 6μg/ml, 7 μg/ml, 8 μg/ml, 9 μg/ml, 10 μg/ml, 11 82 g/ml, 12 μg/ml, 13μg/ml, 14 μg/ml, 15 μg/ml, 16 μg/ml, 17 μg/ml, 18 μg/ml, 19 μg/ml, or 20μg/ml. Preferably, the concentration of psoralen is about 10 μg/ml. Asused herein, the term “about” takes into account that measurements oftime, chemical concentration, temperature, pH, and other factorstypically measured in a laboratory or production facility are neverexact, and may vary by a given amount based on the type of measurementand the instrumentation used to make the measurement.

[0142] The incubation with psoralen is typically carried out for aperiod of time prior to UV exposure. This time period preferably rangesfrom about one minute to about 20 minutes prior to the UV exposure.Preferably, the time period ranges from about 2 minutes to about 19minutes, from about 3 minutes to about 18 minutes, from about 4 minutesto about 17 minutes, from about 5 minutes to about 16 minutes, fromabout 6 minutes to about 15 minutes, from about 7 minutes to about 14minutes, from about 8 minutes to about 13 minutes, or from about 9minutes to about 12 minutes. Accordingly, the incubation time may beabout 1 minute , about 2 minutes, about three minutes, about 4 minutes,about 5 minutes, about 6 minutes , about 7 minutes , about 8 minutes,about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes,about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes,about 17 minutes, about 18 minutes, about 19 minutes, or about 20minutes. More preferably, the incubation is carried out for 10 minutesprior to the UV exposure.

[0143] The psoralen-treated viruses are then exposed to UV light. The UVmay be of any wavelength, but is preferably long-wave UV light, e.g.,about 365 nm. Exposure to UV is carried out for a time period rangingfrom about 0.1 minute to about 20 minutes. Preferably, the time periodranges from about 0.2 minute to about 19 minutes, from about 0.3 minuteto about 18 minutes, from about 0.4 minute to about 17 minutes, fromabout 0.5 minute to about 16 minutes, from about 0.6 minute to about 15minutes, from about 0.7 minute to about 14 minutes, from about 0.8minute to about 13 minutes, from about 0.9 minute to about 12 minutesfrom about 1 minute to about 11 minutes, from about 2 minutes to about10 minutes, from about 2.5 minutes to about 9 minutes, from about 3minutes to about 8 minutes, from about 4 minutes to about 7 minutes, orfrom about 4.5 minutes to about 6 minutes. Accordingly, the incubationtime may be about 0.1 minute, about 0.5 minute, about 1 minute, about 2minutes, about three minutes, about 4 minutes, about 5 minutes, about 6minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10minutes, about 1 1 minutes, about 12 minutes, about 13 minutes, about 14minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18minutes, about 19 minutes, or about 20 minutes. More preferably, thevirus vector is exposed to UV light for a period of about 5 minutes.

[0144] The ability to assemble and express immunoglobulin molecules orantigen-specific fragments thereof in eukaryotic cells from twolibraries of polynucleotides encoding immunoglobulin subunitpolypeptides provides a significant improvement over the methods ofproducing single-chain antibodies in bacterial systems, in that thetwo-step selection process can be the basis for selection ofimmunoglobulin molecules or antigen-specific fragments thereof with avariety of specificities.

[0145] Examples of specific embodiments which further illustrate, but donot limit this embodiment, are provided in the Examples below. Asdescribed in detail, supra, selection of specific immunoglobulin subunitpolypeptides, e.g., immunoglobulin heavy and light chains, isaccomplished in two phases. First, a library of diverse heavy chainsfrom immunoglobulin producing cells of either naive or immunized donorsis constructed in a eukaryotic virus vector, for example, a poxvirusvector, and a similarly diverse library of immunoglobulin light chainsis constructed either in a plasmid vector, in which expression of therecombinant gene is regulated by a virus promoter, or in a eukaryoticvirus vector which has been inactivated, e.g., through psoralen and UVtreatment. Host cells capable of expressing immunoglobulin molecules, orantigen-specific fragments thereof, are infected with virus vectorencoding the heavy chain library at a multiplicity of infection of about1 (MOI=1). “Multiplicity of infection” refers to the average number ofvirus particles available to infect each host cell. For example, if anMOI of 1, i.e., an infection where, on average, each cell is infected byone virus particle, is desired, the number of infectious virus particlesto be used in the infection is adjusted to be equal to the number ofcells to be infected.

[0146] According to this strategy, host cells are either transfectedwith the light chain plasmid library, or infected with the inactivatedlight chain virus library under conditions which allow, on average, 10or more separate polynucleotides encoding light chain polypeptides to betaken up and expressed in each cell. Under these conditions, a singlehost cell can express multiple immunoglobulin molecules, or fragmentsthereof, with different light chains associated with the same heavychains in characteristic H₂L₂ structures in each host cell.

[0147] It will be appreciated by those of ordinary skill in the art thatcontrolling the number of plasmids taken up by a cell is difficult,because successful transfection depends on inducing a competent state incells which may not be uniform and could lead to taking up variableamounts of DNA. Accordingly, in those embodiments where it is desired tocarefully control the number of polynucleotides from the second librarywhich are introduced into each infected host cell, the use of aninactivated virus vector is preferred, because the multiplicity ofinfection of viruses is more easily controlled.

[0148] The expression of multiple light chains in a single host cell,associated with a single heavy chain, has the effect of reducing theavidity of specific antigen immunoglobulin, but may be beneficial forselection of relatively high affinity binding sites. As used herein, theterm “affinity” refers to a measure of the strength of the binding of anindividual epitope with the CDR of an immunoglobulin molecule. See,e.g., Harlow at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen. See, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valencies of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. As will be appreciated by those ofordinary skill in the art, if a host cell expresses immunoglobulinmolecules on its surface, each comprising a given heavy chain, but wheredifferent immunoglobulin molecules on the surface comprise differentlight chains, the “avidity” of that host cell for a given antigen willbe reduced. However, the possibility of recovering a group ofimmunoglobulin molecules which are related in that they comprise acommon heavy chain, but which, through association with different lightchains, react with a particular antigen with a spectrum of affinities,is increased. Accordingly, by adjusting the number of different lightchains, or fragments thereof, which are allowed to associate with acertain number of heavy chains, or fragments thereof in a given hostcell, the present invention provides a method to select for and enrichfor immunoglobulin molecules, or antigen-specific fragments thereof,with varied affinity levels.

[0149] In utilizing this strategy in the first step of the method forselecting immunoglobulin molecules, or antigen-specific fragmentsthereof as described above, the first library is preferably constructedin a eukaryotic virus vector, and the host cells are infected with thefirst library at an MOI ranging from about 1 to about 10, preferablyabout 1, while the second library is introduced under conditions whichallow up to 20 polynucleotides of said second library to be taken up byeach infected host cell. For example, if the second library isconstructed in an inactivated virus vector, the host cells are infectedwith the second library at an MOI ranging from about 1 to about 20,although MOIs higher or lower than this range may be desirable dependingon the virus vector used and the characteristics of the immunoglobulinmolecules desired. If the second library is constructed in a plasmidvector, transfection conditions are adjusted to allow anywhere from 0plasmids to about 20 plasmids to enter each host cell. Selection forlower or higher affinity responses to antigen is controlled byincreasing or decreasing the average number of polynucleotides of thesecond library allowed to enter each infected cell.

[0150] More preferably, where the first library is constructed in avirus vector, host cells are infected with the first library at an MOIranging from about 1-9, about 1-8, about 1-7, about 1-6, about 1-5,about 1-4, or about 1-2. In other words, host cells are infected withthe first library at an MOI of about 10, about 9, about 8, about 7,about 6, about 5, about 4, about 3, about 2, or about 1. Mostpreferably, host cells are infected with the first library at an MOI ofabout 1.

[0151] Where the second library is constructed in a plasmid vector, theplasmid vector is more preferably introduced into host cells underconditions which allow up to about 19, about 18, about 17, about 16,about 15, about 14, about 13, about 12, about 10, about 9, about 8,about 7, about 6, about 5, about 4, about 3 about 2, or about 1polynucleotide(s) of the second library to be taken up by each infectedhost cell. Most preferably, where the second library is constructed in aplasmid vector, the plasmid vector is introduced into host cells underconditions which allow up to about 10 polynucleotides of the secondlibrary to be taken up by each infected host cell.

[0152] Similarly, where the second library is constructed in aninactivated virus vector, it is more preferred to introduce the secondlibrary into host cells at an MOI ranging from about 1-19, about 2-18,about 3-17, about 4-16, about 5-15, about 6-14, about 7 -13, about 8-12,or about 9-11. In other words, host cells are infected with the secondlibrary at an MOI of about 20, about 19, about 18, about 17, about 16,about 15, about 14, about 13, about 12, about 11, about 10, about 9,about 8, about 7, about 6, about 5, about 4, about 3, about 2, orabout 1. In a most preferred aspect, host cells are infected with thesecond library at an MOI of about 10. As will be understood by those ofordinary skill in the art, the titer, and thus the “MOI” of aninactivated virus cannot be directly measured, however, the titer may beinferred from the titer of the starting infectious virus stock which wassubsequently inactivated.

[0153] In a most preferred aspect, the first library is constructed in avirus vector and the second library is constructed in a virus vectorwhich has been inactivated, the host cells are infected with said firstlibrary at an MOI of about 1, and the host cells are infected with thesecond library at an MOI of about 10.

[0154] In the present invention, a preferred virus vector is derivedfrom a poxvirus, e.g., vaccinia virus. If the first library encoding thefirst immunoglobulin subunit polypeptide is constructed in a poxvirusvector and the expression of second immunoglobulin subunit polypeptides,encoded by the second library constructed either in a plasmid vector oran inactivated virus vector, are regulated by a poxvirus promoter, highlevels of the second immunoglobulin subunit polypeptide are expressed inthe cytoplasm of the poxvirus infected cells without a requirement fornuclear integration.

[0155] In the second step of the immunoglobulin selection as describedabove, the second library is preferably constructed in an infectiouseukaryotic virus vector, and the host cells are infected with the secondlibrary at an MOI ranging from about 1 to about 10. More preferably,where the second library is constructed in a virus vector, host cellsare infected with the second library at an MOI ranging from about 1-9,about 1-8, about 1-7, about 1-6, about 1-5, about 1-4, or about 1-2. Inother words, host cells are infected with the second library at an MOIof about 10, about 9, about 8, about 7, about 6, about 5, about 4, about3, about 2, or about 1. Most preferably, host cells are infected withthe second library at an MOI of about 1.

[0156] In the second step of the immunoglobulin selection,polynucleotides from the first library have been isolated. In certainembodiments, a single first library polynucleotide, i.e., a clone, isintroduced into the host cells used to isolate polynucleotides from thesecond library. In this situation, the polynucleotides isolated from thefirst library are introduced into host cells under conditions whichallow at least about 1 polynucleotide per host cell. However, since allthe polynucleotides being introduced from the first library will be thesame, i.e., copies of a cloned polynucleotide, the number ofpolynucleotides introduced into any given host cell is less important.For example, if a cloned polynucleotide isolated from the first libraryis contained in an inactivated virus vector, that vector would beintroduced at an MOI of about 1, but an MOI greater than 1 would beacceptable. Similarly, if a cloned polynucleotide isolated from thefirst library is introduced in a plasmid vector, the number of plasmidswhich are introduced into any given host cell is of little importance,rather, transfection conditions should be adjusted to insure that atleast one polynucleotide is introduced into each host cell. Analternative embodiment may be utilized if, for example, severaldifferent polynucleotides were isolated from the first library. In thisembodiment, pools of two or more different polynucleotides isolated fromthe first library may be advantageously introduced into host cellsinfected with the second library of polynucleotides. In this situation,if the polynucleotides isolated from the first library are contained inan inactivated virus vector, an MOI of inactivated virus particles ofgreater than about 1, e.g., about 2, about 3, about 4, about 5, or moremay be preferred, of if the polynucleotides isolated from the firstlibrary are contained in a plasmid vector, conditions which allow atleast about 2, 3, 4, 5, or more polynucleotides to enter each cell, maybe preferred.

[0157] Poxvirus Vectors. As noted above, a preferred virus vector foruse in the present invention is a poxvirus vector. “Poxvirus” includesany member of the family Poxviridae, including the subfamililesChordopoxviridae (vertebrate poxviruses) and Entomopoxviridae (insectpoxviruses). See, for example, B. Moss in: Virology, 2d Edition, B. N.Fields, D. M. Knipe et al., Eds., Raven Press, p. 2080 (1990). Thechordopoxviruses comprise, inter alia, the following genera:Orthopoxvirus (e.g., vaccinia, variola virus, raccoon poxvirus);Avipoxvirus (e.g., fowlpox); Capripoxvirus (e.g, sheeppox)Leporipoxvirus (e.g., rabbit (Shope) fibroma, and myxoma); andSuipoxvirus (e.g., swinepox). The entomopoxviruses comprise threegenera: A, B and C. In the present invention, orthopoxviruses arepreferred. Vaccinia virus is the prototype orthopoxvirus, and has beendeveloped and is well-characterized as a vector for the expression ofheterologous proteins. In the present invention, vaccinia virus vectors,particularly those that have been developed to perform trimolecularrecombination, are preferred. However, other orthopoxviruses, inparticular, raccoon poxvirus have also been developed as vectors and insome applications, have superior qualities.

[0158] Poxviruses are distinguished by their large size and complexity,and contain similarly large and complex genomes. Notably, poxvirusesreplication takes place entirely within the cytoplasm of a host cell.The central portions of poxvirus genomes are similar, while the terminalportions of the virus genomes are characterized by more variability.Accordingly, it is thought that the central portion of poxvirus genomescarry genes responsible for essential functions common to allpoxviruses, such as replication. By contrast, the terminal portions ofpoxvirus genomes appear responsible for characteristics such aspathogenicity and host range, which vary among the different poxviruses,and may be more likely to be non-essential for virus replication intissue culture. It follows that if a poxvirus genome is to be modifiedby the rearrangement or removal of DNA fragments or the introduction ofexogenous DNA fragments, the portion of the naturally-occurring DNAwhich is rearranged, removed, or disrupted by the introduction ofexogenous DNA is preferably in the more distal regions thought to benon-essential for replication of the virus and production if infectiousvirions in tissue culture.

[0159] The naturally-occurring vaccinia virus genome is a cross-linked,double stranded linear DNA molecule, of about 186,000 base pairs (bp),which is characterized by inverted terminal repeats. The genome ofvaccinia virus has been completely sequenced, but the functions of mostgene products remain unknown. Goebel, S. J., et al., Virology179:247-266,517-563 (1990); Johnson, G. P., et al., Virology196:381-401. A variety of non-essential regions have been identified inthe vaccinia virus genome. See, e.g., Perkus, M. E., et al., Virology152:285-97 (1986); and Kotwal, G. J. and Moss B., Virology 167:524-37.

[0160] In those embodiments where poxvirus vectors, in particularvaccinia virus vectors, are used to express immunglobulin subunitpolypeptides, any suitable poxvirus vector may be used. It is preferredthat the libraries of immunoglobulin subunit polypeptides be carried ina region of the vector which is non-essential for growth and replicationof the vector so that infectious viruses are produced. Although avariety of non-essential regions of the vaccinia virus genome have beencharacterized, the most widely used locus for insertion of foreign genesis the thymidine kinase locus, located in the HindIII J fragment in thegenome. In certain preferred vaccinia virus vectors, the tk locus hasbeen engineered to contain one or two unique restriction enzyme sites,allowing for convenient use of the trimolecular recombination method oflibrary generation. See infra, and also Zauderer, PCT Publication No. WO00/028016.

[0161] Libraries of polynucleotides encoding immunoglobulin subunitpolypeptides are inserted into poxvirus vectors, particularly vacciniavirus vectors, under operable association with a transcriptional controlregion which functions in the cytoplasm of a poxvirus-infected cell.

[0162] Poxvirus transcriptional control regions comprise a promoter anda transcription termination signal. Gene expression in poxviruses istemporally regulated, and promoters for early, intermediate, and lategenes possess varying structures. Certain poxvirus genes are expressedconstitutively, and promoters for these “early-late” genes bear hybridstructures. Synthetic early-late promoters have also been developed. SeeHammond J. M., et al., J. Virol. Methods 66:135-8 (1997); ChakrabartiS., et al., Biotechniques 23:1094-7 (1997). In the present invention,any poxvirus promoter may be used, but use of early, late, orconstitutive promoters may be desirable based on the host cell and/orselection scheme chosen. Typically, the use of constitutive promoters ispreferred.

[0163] Examples of early promoters include the 7.5-kD promoter (also alate promoter), the DNA pol promoter, the tk promoter, the RNA polpromoter, the 19-kD promoter, the 22-kD promoter, the 42-kD promoter,the 37-kD promoter, the 87-kD promoter, the H3′promoter, the H6promoter, the D1 promoter, the D4 promoter, the D5 promoter, the D9promoter, the D12 promoter, the I3 promoter, the M1 promoter, and the N2promoter. See, e.g., Moss, B., “Poxviridae and their Replication” INVirology, 2d Edition, B. N. Fields, D. M. Knipe et al., Eds., RavenPress, p.2088 (1990). Early genes transcribed in vaccinia virus andother poxviruses recognize the transcription termination signal TTTTTNT,where N can be any nucleotide. Transcription normally terminatesapproximately 50 bp upstream of this signal. Accordingly, ifheterologous genes are to be expressed from poxvirus early promoters,care must be taken to eliminate occurrences of this signal in the codingregions for those genes. See, e.g., Earl, P. L., et al., J. Virol.64:2448-51 (1990).

[0164] Example of late promoters include the 7.5-kD promotor, the MILpromoter, the 37-kD promoter, the 11-kD promotor, the 11L promoter, the12L promoter, the 13L promoter, the 15L promoter, the 17L promoter, the28-kD promoter, the H1L promoter, the H3L promoter, the H5L promoter,the H6L promoter, the H8L promoter, the D11L promoter, the D12Lpromotor, the D13L promoter, the A1L promoter, the A2L promoter, the A3Lpromoter, and the P4b promoter. See, e.g., Moss, B., “Poxviridae andtheir Replication” IN Virology, 2d Edition, B. N. Fields, D. M. Knipe etal., Eds., Raven Press, p. 2090 (1990). The late promoters apparently donot recognize the transcription termination signal recognized by earlypromoters.

[0165] Preferred constitutive promoters for use in the present inventioninclude the synthetic early-late promoters described by Hammond andChakrabarti, the MH-5 early-late promoter, and the 7.5-kD or “p7.5”promoter. Examples utilizing these promoters are disclosed herein.

[0166] As will be discussed in more detail below, certain selection andscreening methods based on host cell death require that the mechanismsleading to cell death occur prior to any cytopathic effect (CPE) causedby virus infection. The kinetics of the onset of CPE in virus-infectedcells is dependent on the virus used, the multiplicity of infection, andthe type of host cell. For example, in many tissue culture linesinfected with vaccinia virus at an MOI of about 1, CPE is notsignificant until well after 48 to 72 hours post-infection. This allowsa 2 to 3 day time frame for high level expression of immunoglobulinmolecules, and antigen-based selection independent of CPE caused by thevector. However, this time frame may not be sufficient for certainselection methods, especially where higher MOIs are used, and further,the time before the onset of CPE may be shorter in a desired cell line.There is, therefore, a need for virus vectors, particularly poxvirusvectors such as vaccinia virus, with attenuated cytopathic effects sothat, wherever necessary, the time frame of selection can be extended.

[0167] For example, certain attenuations are achieved through geneticmutation. These may be fully defective mutants, i.e., the production ofinfectious virus particles requires helper virus, or they may beconditional mutants, e.g., temperature sensitive mutants. Conditionalmutants are particularly preferred, in that the virus-infected hostcells can be maintained in a non-permissive environment, e.g., at anon-permissive temperature, during the period where host gene expressionis required, and then shifted to a permissive environment, e.g., apermissive temperature, to allow virus particles to be produced.Alternatively, a fully infectious virus may be “attenuated” by chemicalinhibitors which reversibly block virus replication at defined points inthe infection cycle. Chemical inhibitors include, but are not limited tohydroxyurea and 5-fluorodeoxyuridine. Virus-infected host cells aremaintained in the chemical inhibitor during the period where host geneexpression is required, and then the chemical inhibitor is removed toallow virus particles to be produced.

[0168] A number of attenuated poxviruses, in particular vacciniaviruses, have been developed. For example, modified vaccinia Ankara(MVA) is a highly attenuated strain of vaccinia virus that was derivedduring over 570 passages in primary chick embryo fibroblasts (Mayr, A.et al., Infection 3:6-14 (1975)). The recovered virus deletedapproximately 15% of the wild type vaccinia DNA which profoundly affectsthe host range restriction of the virus. MVA cannot replicate orreplicates very inefficiently in most mammalian cell lines. A uniquefeature of the host range restriction is that the block innon-permissive cells occurs at a relatively late stage of thereplication cycle. Expression of viral late genes is relativelyunimpaired but virion morphogenesis is interrupted (Suter, G. and Moss,B., Proc Natl Acad Sci USA 89:10847-51(1992); Carroll, M. W. and Moss,B., Virology 238:198-211 (1997)). The high levels of viral proteinsynthesis even in non-permissive host cells make MVA an especially safeand efficient expression vector. However, because MVA cannot completethe infectious cycle in most mammalian cells, in order to recoverinfectious virus for multiple cycles of selection it will be necessaryto complement the MVA deficiency by coinfection or superinfection with ahelper virus that is itself deficient and that can be subsequentlyseparated from infectious MVA recombinants by differential expansion atlow MOI in MVA permissive host cells.

[0169] Poxvirus infection can have a dramatic inhibitory effect on hostcell protein and RNA synthesis. These effects on host gene expressioncould, under some conditions, interfere with the selection of specificpoxvirus recombinants that have a defined physiological effect on thehost cell. Some strains of vaccinia virus that are deficient in anessential early gene have been shown to have greatly reduced inhibitoryeffects on host cell protein synthesis. Attenuated poxviruses which lackdefined essential early genes have also been described. See, e.g., U.S.Pat. Nos. 5,766,882, and 5,770,212, by Falkner, et al. Examples ofessential early genes which may be rendered defective include, but arenot limited to the vaccinia virus 17L, F18R, D13L, D6R, A8L, J1R, E7L,F11L, E4L, I1L, J3R, J4R, H7R, and A6R genes. A preferred essentialearly gene to render defective is the D4R gene, which encodes a uracilDNA glycosylase enzyme. Vaccinia viruses defective in defined essentialgenes are easily propagated in complementing cell lines which providesthe essential gene product.

[0170] As used herein, the term “complementation” refers to arestoration of a lost function in trans by another source, such as ahost cell, transgenic animal or helper virus. The loss of function iscaused by loss by the defective virus of the gene product responsiblefor the function. Thus, a defective poxvirus is a non-viable form of aparental poxvirus, and is a form that can become viable in the presenceof complementation. The host cell, transgenic animal or helper viruscontains the sequence encoding the lost gene product, or“complementation element.” The complementation element should beexpressible and stably integrated in the host cell, transgenic animal orhelper virus, and preferably would be subject to little or no risk forrecombination with the genome of the defective poxvirus.

[0171] Viruses produced in the complementing cell line are capable ofinfecting non-complementing cells, and further are capable of high-levelexpression of early gene products. However, in the absence of theessential gene product, host shut-off, DNA replication, packaging, andproduction of infectious virus particles does not take place.

[0172] In particularly preferred embodiments described herein, selectionof desired target gene products expressed in a complex libraryconstructed in vaccinia virus is accomplished through coupling inductionof expression of the complementation element to expression of thedesired target gene product. Since the complementation element is onlyexpressed in those host cells expressing the desired gene product, onlythose host cells will produce infectious virus which is easilyrecovered.

[0173] The preferred embodiments relating to vaccinia virus may bemodified in ways apparent to one of ordinary skill in the art for usewith any poxvirus vector. In the direct selection method, vectors otherthan poxvirus or vaccinia virus may be used.

[0174] The Tri-Molecular Recombination Method. Traditionally, poxvirusvectors such as vaccinia virus have not been used to identify previouslyunknown genes of interest from a complex libraries because a highefficiency, high titer-producing method of constructing and screeninglibraries did not exist for vaccinia. The standard methods ofheterologous protein expression in vaccinia virus involve in vivohomologous recombination and in vitro direct ligation. Using homologousrecombination, the efficiency of recombinant virus production is in therange of approximately 0.1% or less. Although efficiency of recombinantvirus production using direct ligation is higher, the resulting titer isrelatively low. Thus, the use of vaccinia virus vector has been limitedto the cloning of previously isolated DNA for the purposes of proteinexpression and vaccine development.

[0175] Tri-molecular recombination, as disclosed in Zauderer, PCTPublication No. WO 00/028016, is a novel, high efficiency, hightiter-producing method for cloning in vaccinia virus. Using thetri-molecular recombination method, the present inventor has achievedgeneration of recombinant viruses at efficiencies of at least 90%, andtiters at least at least 2 orders of magnitude higher than thoseobtained by direct ligation.

[0176] Thus, in a preferred embodiment, libraries of polynucleotidescapable of expressing immunoglobulin subunit polypeptides areconstructed in poxvirus vectors, preferably vaccinia virus vectors, bytri-molecular recombination.

[0177] By “tri-molecular recombination” or a “tri-molecularrecombination method” is meant a method of producing a virus genome,preferably a poxvirus genome, and even more preferably a vaccinia virusgenome comprising a heterologous insert DNA, by introducing twononhomologous fragments of a virus genome and a transfer vector ortransfer DNA containing insert DNA into a recipient cell, and allowingthe three DNA molecules to recombine in vivo. As a result of therecombination, a viable virus genome molecule is produced whichcomprises each of the two genome fragments and the insert DNA. Thus, thetri-molecular recombination method as applied to the present inventioncomprises: (a) cleaving an isolated virus genome, preferably a DNA virusgenome, more preferably a linear DNA virus genome, and even morepreferably a poxvirus or vaccinia virus genome, to produce a first viralfragment and a second viral fragment, where the first viral fragment isnonhomologous with the second viral fragment; (b) providing a populationof transfer plasmids comprising polynucleotides which encodeimmunoglobulin subunit polypeptides, e.g., immunoglobulin light chains,immunoglobulin heavy chains, or antigen-specific fragments of either,through operable association with a transcription control region,flanked by a 5′ flanking region and a 3′ flanking region, wherein the 5′flanking region is homologous to said the viral fragment described in(a), and the 3′ flanking region is homologous to said second viralfragment described in (a); and where the transfer plasmids are capableof homologous recombination with the first and second viral fragmentssuch that a viable virus genome is formed; (c) introducing the transferplasmids described in (b) and the first and second viral fragmentsdescribed in (a) into a host cell under conditions where a transferplasmid and the two viral fragments undergo in vivo homologousrecombination, i.e., trimolecular recombination, thereby producing aviable modified virus genome comprising a polynucleotide which encodesan immunoglobulin subunit polypeptide; and (d) recovering modified virusgenomes produced by this technique. Preferably, the recovered modifiedvirus genome is packaged in an infectious viral particle.

[0178] By “recombination efficiency” or “efficiency of recombinant virusproduction” is meant the ratio of recombinant virus to total virusproduced during the generation of virus libraries of the presentinvention. As shown in Example 5, the efficiency may be calculated bydividing the titer of recombinant virus by the titer of total virus andmultiplying by 100%. For example, the titer is determined by plaqueassay of crude virus stock on appropriate cells either with selection(e.g., for recombinant virus) or without selection (e.g., forrecombinant virus plus wild type virus). Methods of selection,particularly if heterologous polynucleotides are inserted into the viralthymidine kinase (tk) locus, are well-known in the art and includeresistance to bromdeoxyuridine (BDUR) or other nucleotide analogs due todisruption of the tk gene. Examples of selection methods are describedherein.

[0179] By “high efficiency recombination” is meant a recombinationefficiency of at least 1%, and more preferably a recombinationefficiency of at least about 2%, 2.5%, 3%, 3.5%, 4%, 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.

[0180] A number of selection systems may be used, including but notlimited to the thymidine kinase such as herpes simplex virus thymidinekinase (Wigler, et al., 1977, Cell 11:223), hypoxanthine-guaninephosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl.Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy, etal., 1980, Cell 22:817) genes which can be employed in tk⁻, hgprt⁻ oraprt⁻ cells, respectively. Also, antimetabolite resistance can be usedas the basis of selection for the following genes: dhfr, which confersresistance to methotrexate (Wigler, et al., 1980, Proc. Natl. Acad. Sci.USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527);gpt, which confers resistance to mycophenolic acid (Mulligan & Berg,1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistanceto the aminoglycoside G-418 (Colberre-Garapin, et al., 1981, J. Mol.Biol. 150:1); and hygro, which confers resistance to hygromycin(Santerre, et al., 1984, Gene 30:147).

[0181] Together, the first and second viral fragments or “arms” of thevirus genome, as described above, preferably contain all the genesnecessary for viral replication and for production of infectious viralparticles. Examples of suitable arms and methods for their productionusing vaccinia virus vectors are disclosed herein. See also Falkner etal., U.S. Pat. No. 5,770,212 for guidance concerning essential regionsfor vaccinia replication.

[0182] However, naked poxvirus genomic DNAs such as vaccinia virusgenomes cannot produce infectious progeny without virus-encoded proteinprotein(s)/function(s) associated with the incoming viral particle. Therequired virus-encoded functions, include an RNA polymerase thatrecognizes the transfected vaccinia DNA as a template, initiatestranscription and, ultimately, replication of the transfected DNA. SeeDorner, et al. U.S. Pat. No. 5,445,953.

[0183] Thus, to produce infectious progeny virus by trimolecularrecombination using a poxvirus such as vaccinia virus, the recipientcell preferably contains packaging function. The packaging function maybe provided by helper virus, i.e., a virus that, together with thetransfected naked genomic DNA, provides appropriate proteins and factorsnecessary for replication and assembly of progeny virus.

[0184] The helper virus may be a closely related virus, for instance, apoxvirus of the same poxvirus subfamily as vaccinia, whether from thesame or a different genus. In such a case it is advantageous to select ahelper virus which provides an RNA polymerase that recognizes thetransfected DNA as a template and thereby serves to initiatetranscription and, ultimately, replication of the transfected DNA. If aclosely related virus is used as a helper virus, it is advantageous thatit be attenuated such that formation of infectious virus will beimpaired. For example, a temperature sensitive helper virus may be usedat the non-permissive temperature. Preferably, a heterologous helpervirus is used. Examples include, but are not limited to an avipox virussuch as fowlpox virus, or an ectromelia virus (mouse pox) virus. Inparticular, avipoxviruses are preferred, in that they provide thenecessary helper functions, but do not replicate, or produce infectiousvirions in mammalian cells (Scheiflinger, et al., Proc. Natl. Acad. Sci.USA 89:9977-9981 (1992)). Use of heterologous viruses minimizesrecombination events between the helper virus genome and the transfectedgenome which take place when homologous sequences of closely relatedviruses are present in one cell. See Fenner & Comben, Virology 5:530(1958); Fenner, Virology 8:499 (1959).

[0185] Alternatively, the necessary helper functions in the recipientcell is supplied by a genetic element other than a helper virus. Forexample, a host cell can be transformed to produce the helper functionsconstitutively, or the host cell can be transiently transfected with aplasmid expressing the helper functions, infected with a retrovirusexpressing the helper functions, or provided with any other expressionvector suitable for expressing the required helper virus function. SeeDorner, et al. U.S. Pat. No. 5,445,953.

[0186] According to the trimolecularrecombination method, the first andsecond viral genomic fragments are unable to ligate or recombine witheach other, i.e., they do not contain compatible cohesive ends orhomologous regions, or alternatively, cohesive ends have been treatedwith a dephosphorylating enzyme. In a preferred embodiment, a virusgenome comprises a first recognition site for a first restrictionendonuclease and a second recognition site for a second restrictionendonuclease, and the first and second viral fragments are produced bydigesting the viral genome with the appropriate restrictionendonucleases to produce the viral “arms,” and the first and secondviral fragments are isolated by standard methods. Ideally, the first andsecond restriction endonuclease recognition sites are unique in theviral genome, or alternatively, cleavage with the two restrictionendonucleases results in viral “arms” which include the genes for allessential functions, i.e., where the first and second recognition sitesare physically arranged in the viral genome such that the regionextending between the first and second viral fragments is not essentialfor virus infectivity.

[0187] In a preferred embodiment where a vaccinia virus vector is usedin the trimolecular recombination method, a vaccinia virus vectorcomprising a virus genome with two unique restriction sites within thetk gene is used. In certain preferred vaccinia virus genomes, the firstrestriction enzyme is NotI, having the recognition site GCGGCCGC in thetk gene, and the second restriction enzyme is ApaI, having therecognition site GGGCCC in the tk gene. Even more preferred are vacciniavirus vectors comprising a v7.5/tk virus genome or a vEL/tk virusgenome.

[0188] According to this embodiment, a transfer plasmid with flankingregions capable of homologous recombination with the region of thevaccinia virus genome containing the thymidine kinase gene is used. Afragment of the vaccinia virus genome comprising the HindIII-J fragment,which contains the tk gene, is conveniently used.

[0189] Where the virus vector is a poxvirus, the insert polynucleotidesare preferably operably associated with poxvirus expression controlsequences, more preferably, strong constitutive poxvirus promoters suchas p7.5 or a synthetic early/late promoter.

[0190] Accordingly, a transfer plasmid of the present inventioncomprises a polynucleotide encoding an immunoglobulin subunitpolypeptide, e.g., an heavy chain, and immunoglobulin light chain, or anantigen-specific fragment of a heavy chain or a light chain, throughoperable association with a vaccinia virus p7.5 promoter, or a syntheticearly/late promoter.

[0191] A preferred transfer plasmid of the present invention whichcomprises a polynucleotide encoding an immunoglobulin heavy chainpolypeptide through operable association with a vaccinia virus p7.5promoter is pVHE, which comprises the sequence:GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCAAACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGCGCATATGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAACCCTTTTCCCCCTCGTCTCCTGTGAGAATTCCCCGTCGGATACGAGCAGCGTGGCCGTTGGCTGCCTCGCACAGGACTTCCTTCCCGACTCCATCACTTTCTCCTGGAAATACAAGAACAACTCTGACATCAGCAGCACCCGGGGCTTCCCATCAGTCCTGAGAGGGGGCAAGTACGCAGCCACCTCACAGGTGCTGCTGCCTTCCAAGGACGTCATGCAGGGCACAGACGAACACGTGGTGTGCAAAGTCCAGCACCCCAACGGCAACAAAGAAAAGAACGTGCCTCTTCCAGTGATTGCTGAGCTGCCTCCCAAAGTGAGCGTCTTCGTCCCACCCCGCGACGGCTTCTTCGGCAACCCCCGCAGCAAGTCCAAGCTCATCTGCCAGGCCACGGGTTTCAGTCCCCGGCAGATTCAGGTGTCCTGGCTGCGCGAGGGGAAGCAGGTGGGGTCTGGCGTCACCACGGACCAGGTGCAGGCTGAGGCCAAAGAGTCTGGGCCCACGACCTACAAGGTGACTAGCACACTGACCATCAAAGAGAGCGACTGGCTCAGCCAGAGCATGTTCACCTGCCGCGTGGATCACAGGGGCCTGACCTTCCAGCAGAATGCGTCCTCCATGTGTGTCCCCGATCAAGACACAGCCATCCGGGTCTTCGCCATCCCCCCATCCTTTGCCAGCATCTTCCTCACCAAGTCCACCAAGTTGACCTGCCTGGTCACAGACCTGACCACCTATGACAGCGTCACCATCTCCTGGACCCGCCAGAATGGCGAAGCTGTGAAAACCCACACCAACATCTCCGAGAGCCACCCCAATGCCACTTTCAGCGCCGTGGGTGAGGCCAGCATCTGCGAGGATGACTGGAATTCCGGGGAGAGGTTCACGTGCACCGTGACCCACACAGACCTGCCCTCGCCACTGAAGCAGACCATCTCCCGGCCCAAGGGGGTGGCCCTGCACAGGCCCGATGTCTACTTGCTGCCACCAGCCCGGGAGCAGCTGAACCTGCGGGAGTCGGCCACCATCACGTGCCTGGTGACGGGCTTCTCTCCCGCGGACGTCTTCGTGCAGTGGATGCAGAGGGGGCAGCCCTTGTCCCCGGAGAAGTATGTGACCAGCGCCCCAATGCCTGAGCCCCAGGCCCCAGGCCGGTACTTCGCCCACAGCATCCTGACCGTGTCCGAAGAGGAATGGAACACGGGGGAGACCTACACCTGCGTGGTGGCCCATGAGGCCCTGCCCAACAGGGTCACTGAGAGGACCGTGGACAAGTCCACCGAGGGGGAGGTGAGCGCCGACGAGGAGGGCTTTGAGAACCTGTGGGCCACCGCCTCCACCTTCATCGTCCTCTTCCTCCTGAGCCTCTTCTACAGTACCACCGTCACCTTGTTCAAGGTGAAATGAGTCGAC

[0192] designated herein as SEQ ID NO:14. PCR-amplified heavy chainvariable regions may be inserted in-frame into unique BssHII (atnucleotides 96-100 of SEQ ID NO:15), and B stEII (nucleotides 106-112 ofSEQ ID NO:16) sites, which are indicated above in bold.

[0193] Furthermore, pVHE may be used in those embodiments where it isdesired to transfer polynucleotides isolated from the first library intoa plasmid vector for subsequent selection of polynucleotides of thesecond library as described above.

[0194] Another preferred transfer plasmid of the present invention whichcomprises a polynucleotide encoding an immunoglobulin kappa light chainpolypeptide through operable association with a vaccinia virus p7.5promoter is pVKE, which comprises the sequence:GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACACGGGTGCACTTGACTCGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGG TCGAC

[0195] designated herein as SEQ ID NO:17. PCR-amplified kappa lightchain variable regions may be inserted in-frame into unique ApaLI(nucleotides 95-100 of SEQ ID NO:18), and XhoI (nucleotides 105-110 ofSEQ ID NO:19) sites, which are indicated above in bold.

[0196] Furthermore, pVKE may be used in those embodiments where it isdesired to have polynucleotides of the second library in a a plasmidvector during the selection of polynucleotides of the first library asdescribed above.

[0197] Another preferred transfer plasmid of the present invention whichcomprises a polynucleotide encoding an immunoglobulin lambda light chainpolypeptide through operable association with a vaccinia virus p7.5promoter is pVLE, which comprises the sequence:GGCCAAAAATTGAAAAACTAGATCTATTTATTGCACGCGGCCGCCCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTTGACTCGAGAAGCTTACCGTCCTACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGTCGAC

[0198] designated herein as SEQ ID NO:20. PCR-amplified lambda lightchain variable regions may be inserted in-frame into unique ApaLI(nucleotides 95-100 of SEQ ID NO:21) and HindIII (nucleotides 111-116 ofSEQ ID NO:22) sites, which are indicated above in bold.

[0199] Furthermore, pVLE maybe used in those embodiments where it isdesired to have polynucleotides of the second library in a a plasmidvector during the selection of polynucleotides of the first library asdescribed above.

[0200] By “insert DNA” is meant one or more heterologous DNA segments tobe expressed in the recombinant virus vector. According to the presentinvention, “insert DNAs” are polynucleotides which encode immunoglobulinsubunit polypeptides. A DNA segment may be naturally occurring, nonnaturally occurring, synthetic, or a combination thereof. Methods ofproducing insert DNAs of the present invention are disclosed herein.

[0201] By “transfer plasmid” is meant a plasmid vector containing aninsert DNA positioned between a 5′ flanking region and a 3′ flankingregion as described above. The 5′ flanking region shares homology withthe first viral fragment, and the 3′ flanking region shares homologywith the second viral fragment. Preferably, the transfer plasmidcontains a suitable promoter, such as a strong, constitutive vacciniapromoter where the virus vector is a poxvirus, upstream of the insertDNA. The term “vector” means a polynucleotide construct containing aheterologous polynucleotide segment, which is capable of effectingtransfer of that polynucleotide segment into a suitable host cell.Preferably the polynucleotide contained in the vector is operably linkedto a suitable control sequence capable of effecting the expression ofthe polynucleotide in a suitable host. Such control sequences include apromoter to effect transcription, an optional operator sequence tocontrol such transcription, a sequence encoding suitable mRNA ribosomebinding sites, and sequences which control the termination oftranscription and translation. As used herein, a vector may be aplasmid, a phage particle, a virus, a messenger RNA, or simply apotential genomic insert. Once transformed into a suitable host, thevector may replicate and function independently of the host genome, ormay in some instances, integrate into the genome itself. Typical plasmidexpression vectors for mammalian cell culture expression, for example,are based on pRK5 (EP 307,247), pSV16B (WO 91/08291) and pVL1392(Pharmingen).

[0202] However, “a transfer plasmid,” as used herein, is not limited toa specific plasmid or vector. Any DNA segment in circular or linear orother suitable form may act as a vehicle for transferring the DNA insertinto a host cell along with the first and second viral “arms” in thetri-molecular recombination method. Other suitable vectors includelambda phage, mRNA, DNA fragments, etc., as described herein orotherwise known in the art. A plurality of plasmids may be a “primarylibrary” such as those described herein for lambda.

[0203] Modifications of Trimolecular Recombination. Trimolecularrecombination can be used to construct cDNA libraries in vaccinia viruswith titers of the order of about 10⁷ pfu. There are several factorsthat limit the complexity of these cDNA libraries or other libraries.These include: the size of the primary cDNA library or other library,such as a library of polynucleotides encoding immunoglobulin subunitpolypeptides, that can be constructed in a plasmid vector, and the laborinvolved in the purification of large quantities (hundreds ofmicrograms) of virus “arms,” preferably vaccinia virus “arms” or otherpoxvirus “arms.” Modifications of trimolecular recombination that wouldallow for vaccinia or other virus DNA recombination with primary cDNAlibraries or other libraries, such as polynucleotides encodingimmunoglobulin subunit polypeptides, constructed in bacteriophage lambdaor DNA or phagemids derived therefrom, or that would allow separatevirus DNA arms to be generated in vivo following infection with amodified viral vector could greatly increase the quality and titer ofthe eukaryotic virus cDNA libraries or other libraries that areconstructed using these methods.

[0204] Transfer of cDNA inserts from a Bacteriophage Lambda Library toVaccinia Virus. Lambda phage vectors have several advantages overplasmid vectors for construction of cDNA libraries or other libraries,such as polynucleotides encoding immunoglobulin subunit polypeptides.Plasmid cDNA (or other DNA insert) libraries or linear DNA libraries areintroduced into bacteria cells by chemical/heat shock transformation, orby electroporation. Bacteria cells are preferentially transformed bysmaller plasmids, resulting in a potential loss of representation oflonger cDNAs or other insert DNA, such as polynucleotides encodingimmunoglobulin subunit polypeptides, in a library. In addition,transformation is a relatively inefficient process for introducingforeign DNA or other DNA into a cell requiring the use of expensivecommercially prepared competent bacteria in order to construct a cDNAlibrary or other library, such as polynucleotides encodingimmunoglobulin subunit polypeptides. In contrast, lambda phage vectorscan tolerate cDNA inserts of 12 kilobases or more without any size bias.Lambda vectors are packaged into virions in vitro using high efficiencycommercially available packaging extracts so that the recombinant lambdagenomes can be introduced into bacterial cells by infection. Thisresults in primary libraries with higher titers and betterrepresentation of large cDNAs or other insert DNA, such aspolynucleotides encoding immunoglobulin subunit polypeptides, than iscommonly obtained in plasmid libraries.

[0205] To enable transfer of cDNA inserts or other insert DNA, such aspolynucleotides encoding immunoglobulin subunit polypeptides, from alibrary constructed in a lambda vector to a eukaryotic virus vector suchas vaccinia virus, the lambda vector must be modified to includevaccinia virus DNA sequences that allow for homologous recombinationwith the vaccinia virus DNA. The following example uses vaccinia virushomologous sequences, but other viruses may be similarly used. Forexample, the vaccinia virus HindIII J fragment (comprising the vacciniatk gene) contained in plasmid p7.5/ATG0/tk (as described in Example 5,infra) can be excised using HindIII and SnaBI (3 kb of vaccinia DNAsequence), and subcloned into the HindIII/SnaBI sites of pT7Blue3(Novagen cat no. 70025-3) creating pT7B3.Vtk. The vaccinia tk gene canbe excised from this vector with SacI and SnaBI and inserted into theSacI/SmaI sites of Lambda Zap Express (Stratagene) to create lambda.Vtk.The lambda.Vtk vector will contain unique NotI, BamHI, SmaI, and SalIsites for insertion of cDNA downstream of the vaccinia 7.5 k promoter.cDNA libraries can be constructed in lambda.Vtk employing methods thatare well known in the art.

[0206] DNA from a cDNA library or other library, such as polynucleotidesencoding immunoglobulin subunit polypeptides, constructed in lambda.Vtk,or any similar bacteriophage that includes cDNA inserts or other insertDNA with flanking vaccinia DNA sequences to promote homologousrecombination, can be employed to generate cDNA or other insert DNArecombinant vaccinia virus. Methods are well known in the art forexcising a plasmid from the lambda genome by coinfection with a helperphage (ExAssist phage, Stratagene cat no. 211203). Mass excision from alambda based library creates an equivalent cDNA library or other libraryin a plasmid vector. Plasmids excised from, for example, the lambda.VtkcDNA library will contain the vaccinia tk sequences flanking the cDNAinserts or other insert DNAs, such as polynucleotides encodingimmunoglobulin subunit polypeptides. This plasmid DNA can then be usedto construct vaccinia recombinants by trimolecular recombination.Another embodiment of this method is to purify the lambda DNA directlyfrom the initial lambda.Vtk library, and to transfect this recombinantviral (lambda) DNA or fragments thereof together with the two largevaccinia virus DNA fragments for trimolecular recombination.

[0207] Generation of vaccinia arms in vivo. Purification andtransfection of vaccinia DNA or other virus DNA “arms” or fragments is alimiting factor in the construction of polynucleotide libraries bytrimolecular recombination. Modifications to the method to allow for therequisite generation of virus arms, in particular vaccinia virus arms,in vivo would allow for more efficient construction of libraries ineukaryotic viruses.

[0208] Host cells can be modified to express a restriction endonucleasethat recognizes a unique site introduced into a virus vector genome. Forexample, when a vaccinia virus infects these host cells, the restrictionendonuclease will digest the vaccinia DNA, generating “arms” that canonly be repaired, i.e., rejoined, by trimolecular recombination.Examples of restriction endonucleases include the bacterial enzymes NotIand ApaI, the Yeast endonuclease VDE (R. Hirata, Y. Ohsumi, A. Nakano,H. Kawasaki, K. Suzuki, Y. Anraku. 1990 J. Biological Chemistry 265:6726-6733), the Chlamydomonas eugametos endonuclease I-CeuI and otherswell-known in the art. For example, a vaccinia strain containing uniqueNotI and ApaI sites in the tk gene has already been constructed, and astrain containing unique VDE and/or I-CeuI sites in the tk gene could bereadily constructed by methods known in the art.

[0209] Constitutive expression of a restriction endonuclease would belethal to a cell, due to the fragmentation of the chromosomal DNA bythat enzyme. To avoid this complication, in one embodiment host cellsare modified to express the gene(s) for the restriction endonuclease(s)under the control of an inducible promoter.

[0210] A preferred method for inducible expression utilizes the Tet-OnGene Expression System (Clontech). In this system expression of the geneencoding the endonuclease is silent in the absence of an inducer(tetracycline). This makes it possible to isolate a stably transfectedcell line that can be induced to express a toxic gene, i.e., theendonuclease (Gossen, M. et al., Science 268: 1766-1769 (1995)). Theaddition of the tetracycline derivative doxycycline induces expressionofthe endonuclease. In a preferred embodiment, BSC1 host cells will bestably transfected with the Tet-On vector controlling expression of theNotI gene. Confluent monolayers of these cells will be induced withdoxycycline and then infected with v7.5/tk (unique NotI site in tkgene), and transfected with cDNA or insert DNA recombinant transferplasmids or transfer DNA or lambda phage or phagemid DNA. Digestion ofexposed vaccinia DNA at the unique NotI site, for example, in the tkgene or other sequence by the NotI endonuclease encoded in the hostcells produces two large vaccinia DNA fragments which can give rise tofull-length viral DNA only by undergoing trimolecular recombination withthe transfer plasmid or phage DNA. Digestion of host cell chromosomalDNA by NotI is not expected to prevent production of modified infectiousviruses because the host cells are not required to proliferate duringviral replication and virion assembly.

[0211] In another embodiment of this method to generate virus arms suchas vaccinia arms in vivo, a modified vaccinia strain is constructed thatcontains a unique endonuclease site in the tk gene or othernon-essential gene, and also contains a heterologous polynucleotideencoding the endonuclease under the control of the T7 bacteriophagepromoter at another non-essential site in the vaccinia genome. Infectionof cells that express the T7 RNA polymerase would result in expressionof the endonuclease, and subsequent digestion of the vaccinia DNA bythis enzyme. In a preferred embodiment, the v7.5/tk strain of vacciniais modified by insertion of a cassette containing the cDNA encoding NotIwith expression controlled by the T7 promoter into the HindIII C or Fregion (Coupar, E. H. B. et al., Gene 68: 1-10 (1988); Flexner, C. etal., Nature 330: 259-262 (1987)), generating v7.5/tk/T7NotI. A cell lineis stably transfected with the cDNA encoding the T7 RNA polymerase underthe control of a mammalian promoter as described (O. Elroy-Stein, B.Moss. 1990 Proc. Natl. Acad. Sci. USA 87: 6743-6747). Infection of thispackaging cell line with v7.5/tk/T7NotI will result in T7 RNA polymerasedependent expression of NotI, and subsequent digestion of the vacciniaDNA into arms. Infectious full-length viral DNA can only bereconstituted and packaged from the digested vaccinia DNA arms followingtrimolecular recombination with a transfer plasmid or phage DNA. In yetanother embodiment of this method, the T7 RNA polymerase can be providedby co-infection with a T7 RNA polymerase recombinant helper virus, suchas fowlpox virus (P. Britton, P. Green, S. Kottier, K. L. Mawditt, Z.Penzes, D. Cavanagh, M. A. Skinner. 1996 J. General Virology 77:963-967).

[0212] A unique feature of trimolecular recombination employing thesevarious strategies for generation of large virus DNA fragments,preferably vaccinia DNA fragments in vivo is that digestion of thevaccinia DNA may, but does not need to precede recombination. Itsuffices that only recombinant virus escapes destruction by digestion.This contrasts with trimolecular recombination employing transfection ofvaccinia DNA digested in vitro where, of necessity, vaccinia DNAfragments are created prior to recombination. It is possible thattheopportunity for bimolecular recombination prior to digestion will yielda greater frequency of recombinants than can be obtained throughtrimolecular recombination following digestion.

[0213] Selection and Screening Strategies for Isolation of RecombinantImmunoglobulin Molecules Using Virus Vectors, Especially Poxviruses. Incertain embodiments of the present invention, the trimolecularrecombination method is used in the production of libraries ofpolynucleotides expressing immunoglobulin subunit polypeptides. In thisembodiment, libraries comprising full-length immunoglobulin subunitpolypeptides, or fragments thereof, are prepared by first insertingcassettes encoding immunoglobulin constant regions and signal peptidesinto a transfer plasmid which contains 5′ and 3′ regions homologous tovaccinia virus. Rearranged immunoglobulin variable regions are isolatedby PCR from pre-B cells from unimunized animals of from B cells orplasma cells from immunized animals. These PCR fragments are clonedbetween, and in frame with the immunoglobulin signal peptide andconstant region, to produce a coding region for an immunoglobulinsubunit polypeptide. These transfer plasmids are introduced into hostcells with poxvirus “arms,” and the tri-molecular recombination methodis used to produce the libraries.

[0214] The present invention provides a variety of methods foridentifying, i.e., selecting or screening for immunoglobulin moleculeswith a desired specificity, where the immunoglobulin molecules areproduced in vitro in eukaryotic cells. These include selecting for hostcell effects such as antigen-induced cell death and antigen-inducedsignaling, screening pools of host cells for antigen-specific binding,and screening the medium in which pools of host cells are grown for thepresence of soluble immunoglobulin molecules with a desired antigenicspecificity or a desired functional characteristic.

[0215] As disclosed in detail herein, methods are provided to identifyimmunoglobulin molecules, or antigen-specific fragments thereofexpressed in eukaryotic cells on the basis of either antigen-inducedcell death, antigen-induced signaling, antigen-specific binding, orother antigen-specific functions. The selection and screening techniquesof the present invention eliminate the bias imposed by selection ofantibodies in rodents or the limitations of synthesis and assembly inbacteria.

[0216] Many of the identification methods described herein depend onexpression of host cell genes or host cell transcriptional regulatoryregions, which directly or indirectly induce cell death or produce adetectable signal in response to antigen binding to immunoglobulinmolecules, or antigen-specific fragments thereof, expressed on thesurface of the host cells. It is important to note that most preferredembodiments of the present invention require that host cells be infectedwith a eukaryotic virus vector, preferably a poxvirus vector, and evenmore preferably a vaccinia virus vector. It is well understood by thoseof ordinary skill in the art that some host cell protein synthesis israpidly shut down upon poxvirus infection in some cell lines, even inthe absence of viral gene expression. This is problematic ifupregulation of host cell genes or host cell transcriptional regulatoryregions is required in order to induce antigen-induced cell death orcell signaling. This problem is not intractable, however, because incertain cell lines, inhibition of host protein synthesis remainsincomplete until after viral DNA replication. See Moss, B., “Poxviridaeand their Replication” IN Virology, 2d Edition, B. N. Fields, D. M.Knipe et al., Eds., Raven Press, p. 2096 (1990). There is a need,however, to rapidly screen a variety of host cells for their ability toexpress gene products which are upregulated upon cross linking ofsurface-expressed immunoglobulin molecules upon infection by aeukaryotic virus vector, preferably a poxvirus vector, and even morepreferably a vaccinia virus vector; and to screen desired host cells fordifferential expression of cellular genes upon virus infection withvarious mutant and attenuated viruses.

[0217] Accordingly, a method is provided for screening a variety of hostcells for the expression of host cell genes and/or the operability ofhost cell transcriptional regulatory regions effecting antigen-inducedcell death or cell signaling, upon infection by a virus vector, throughexpression profiling of particular host cells in microarrays of orderedcDNA libraries. Expression profiling in microarrays is described inDuggan, D. J., et al., Nature Genet. 21(1 Suppl):10-14 (1999), which isincorporated herein by reference in its entirety.

[0218] According to this method, expression profiling is used to comparehost cell gene expression patterns in uninfected host cells and hostcells infected with a eukaryotic virus expression vector, preferably apoxvirus vector, even more preferably a vaccinia virus vector, where theparticular eukaryotic virus vector is the vector used to construct saidfirst and said second libraries of polynucleotides of the presentinvention. In this way, suitable host cells capable of expressingimmunoglobulin molecules, or antigen-specific fragments thereof on theirsurface, and which further continue to undergo expression of thenecessary inducible proteins upon infection with a given virus, can beidentified.

[0219] Expression profiling is also used to compare host cell geneexpression patterns in a given host cell, for example, comparingexpression patterns when the host cell is infected with a fullyinfectious virus vector, and when the host cell is infected with acorresponding attenuated virus vector. Expression profiling inmicroarrays allows large-scale screening of host cells infected with avariety of attenuated viruses, where the attenuation is achieved in avariety of different ways. For example, certain attenuations areachieved through genetic mutation. Many vaccinia virus mutants have beencharacterized. These may be fully defective mutants, i.e., theproduction of infectious virus particles requires helper virus, or theymay be conditional mutants, e.g., temperature sensitive mutants.Conditional mutants are particularly preferred, in that thevirus-infected host cells can be maintained in a non-permissiveenvironment, e.g., at a non-permissive temperature, during the periodwhere host gene expression is required, and then shifted to a permissiveenvironment, e.g., a permissive temperature, to allow virus particles tobe produced. Alternatively, a fully infectious virus may be “attenuated”by chemical inhibitors which reversibly block virus replication atdefined points in the infection cycle. Chemical inhibitors include, butare not limited to hydroxyurea and 5-fluorodeoxyuridine. Virus-infectedhost cells are maintained in the chemical inhibitor during the periodwhere host gene expression is required, and then the chemical inhibitoris removed to allow virus particles to be produced.

[0220] Using this method, expression profiling in microarrays may beused to identify suitable host cells, suitable transcription regulatoryregions, and/or suitable attenuated viruses in any of the selectionmethods described herein.

[0221] In one embodiment, a selection method is provided to selectpolynucleotides encoding immunoglobulin molecules, or antigen-specificfragments thereof, based on direct antigen-induced apoptosis. Accordingto this method, a host cell is selected for infection and/ortransfection that is an early B cell lymphoma. Suitable early B celllymphoma cell lines include, but are not limited to CH33 cells, CH31cells (Pennell, C. A., et al., Proc. Natl. Acad. Sci. USA 82:3799-3803(1985)), or WEHI-231 cells (Boyd, A. W. and Schrader, J. W. J. Immunol.126:2466-2469 (1981)). Early B cell lymphoma cell lines respond tocrosslinking of antigen-specific immunoglobulin by induction ofspontaneous growth inhibition and apoptotic cell death (Pennell, C. A.,and Scott, D. W. Eur. J. Immunol. 16:1577-1581 (1986); Tisch, R., etal., Proc. Natl. Acad. Sci. USA 85:69114-6918 (1988); Ales-Martinez, J.E., et al., Proc. Natl. Acad. Sci. USA 85:69119-6923 (1988); Warner, G.L., and Scott, D. W. Cell. Immunol. 115:195-203 (1988)). Followinginfection and/or transfection with the first and second polynucleotidelibraries as described above, synthesis and assembly of antibodymolecules is allowed to proceed for a time period ranging from about 5hours to about 48 hours, preferably for about 6 hours, about 10 hours,about 12 hours, about 16 hours about 20 hours, about 24 hours about 30hours, about 36 hours, about 40 hours, or about 48 hours, even morepreferably for about 12 hours or for about 24 hours; at which time thehost cells are contacted with specific antigen, in order to cross-linkany specific immunoglobulin receptors (i.e., membrane-boundimmunoglobulin molecules, or antigen-specific fragments thereof) andinduce apoptosis in those immunoglobulin expressing host cells whichdirectly respond to cross-linking of antigen-specific immunoglobulin byinduction of growth inhibition and apoptotic cell death. Host cellswhich have undergone apoptosis, or their contents, including thepolynucleotides encoding an immunoglobulin subunit polypeptide which arecontained therein, are recovered, thereby enriching for polynucleotidesof the first library which encode a first immunoglobulin subunitpolypeptide which, as part of an immunoglobulin molecule, orantigen-specific fragment thereof, specifically binds the antigen ofinterest.

[0222] Upon further selection and enrichment steps for polynucleotidesof the first library, and isolation of those polynucleotides, a similarprocess is carried out to recover polynucleotides of the second librarywhich, as part of an immunoglobulin molecule, or antigen-specificfragment thereof, bind the desired specific antigen.

[0223] An example of this method is shown in FIG. 1. A “first library”of polynucleotides encoding diverse heavy chains from antibody producingcells of either naive or immunized donors is constructed in a poxvirusvector, preferably a vaccinia virus vector, and a similarly diverse“second library” of polynucleotides encoding immunoglobulin light chainsis constructed in a plasmid vector in which expression of thepolynucleotides is regulated by a vaccinia promoter, preferably asynthetic early/late promoter, for example the p11 promoter, or the p7.5promoter. Preferably for this embodiment, the immunoglobulin heavy chainconstant region encoded by the poxvirus constructs is designed to retainthe transmembrane region that results in expression of immunoglobulinreceptor on the surface membrane. Eukaryotic cells, preferably early Bcell lymphoma cells, are infected with the pox virus heavy chain libraryat a multiplicity of infection of about 1 (MOI=1). Two hours later theinfected cells are transfected with the light chain plasmid libraryunder conditions which allow, on average, 10 or more separate lightchain recombinant plasmids to be taken up and expressed in each cell.Because expression of the recombinant gene in this plasmid is regulatedby a vaccinia virus promoter, high levels of the recombinant geneproduct are expressed in the cytoplasm of vaccinia virus infected cellswithout a requirement for nuclear integration. In addition, a sequenceindependent mechanism for amplification of circular DNA in the cytoplasmof vaccinia virus infected cell results in even higher concentrations ofthe transfected light chain recombinant plasmids (Merchlinsky, M., andMoss, B. Cancer Cells 6:87-93 (1988). These two factors contribute tothe high levels of expression that result in excess light chainsynthesis.

[0224] Another preferred embodiment utilizes a T7 phage promoter, whichis active in cells in which T7 RNA polymerase is expressed, for theregulation of the expression of polynucleotides encoding a “firstlibrary” of polynucleotides encoding diverse heavy chains from antibodyproducing cells of either naive or immunized donors constructed in apoxvirus vector, preferably a vaccinia virus vector, and a similarlydiverse “second library” of polynucleotides encoding immunoglobulinlight chains is constructed in a plasmid vector (Eckert D. andMerchlinsky M. J Gen Virol. 80 (Pt 6):1463-9 (1999); Elroy-Stein O.,Fuerst T. R. and Moss B. Proc Natl Acad Sci USA. 86(16):6126-30 (1989);Fuerst T. R., Earl P. L. and Moss B. Mol Cell Biol. 7(7):2538-44 (1987);Elroy-Stein O. and Moss B. Proc Natl Acad Sci USA. 87(17):6743-7 (1990);Cottet S. and Corthesy B. Eur J Biochem 246(1):23-31.).

[0225] As will be readily appreciated by those of ordinary skill in theart, kinetic considerations are very important in the design of thisexperiment as the pox virus derived expression vector is itselfcytopathic in a time frame of about 1 to 10 days, more usually about 2to 8 days, 2 to 6 days, or 2 to 4 days, depending on the virus vectorused, the particular host cell, and the multiplicity of infection. In apreferred embodiment, a B cell lymphoma is selected for which theapoptotic response to surface immunoglobulin crosslinking is rapidrelative to the natural cytopathic effects of pox virus infection inthat cell. Accordingly, it is preferred that apoptosis in response toantigen-induced cross-linking of immunoglobulin molecules on the surfaceof the host cells occurs within a period between about 1 hour to about 4days after contacting the host cells with antigen, so as to precedeinduction of CPE. More preferably, apoptosis occurs within about 1 hourabout 2 hours, about 3 hours about 4 hours, about 5 hours, about 6hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,about 11 hours, about 12 hours, about 14 hours, about 16 hours, about 18hours, about 20 hours, about 22 hours, about 24 hours, about 28 hours,about 32 hours, about 36 hours, about 40 hours, aobut 44 hours, or about48 hours after contacting the host cells with antigen. Even morepreferably apoptosis is induced within about 12 hours of contacting thehost cells with antigen. Alternatively, an attenuated poxvirus vector isemployed with a much slower kinetics of induction of cytopathic effects.Attenuated poxvirus vectors are disclosed herein.

[0226] According to this method, host cells which expressantigen-specific immunoglobulins on their surface are selected uponundergoing apoptosis. For example, if the host cells are attached to asolid substrate, those cells which undergo apoptosis are released fromthe substrate and are recovered by harvesting the liquid medium in whichthe host cells are cultured. Alternatively, the host cells are attachedto a solid substrate, and those cells which undergo apoptosis undergo alytic event, thereby releasing their cytoplasmic contents into theliquid medium in which the host cells are cultured. Virus particlesreleased from these cells can then be harvested in the liquid medium.

[0227] A host cell containing a polynucleotide encoding animmunoglobulin subunit polypeptide may become “nonadherent” or“nonviable” by any mechanism, which may include lysis, inability toadhere, loss of viability, loss of membrane integrity, loss ofstructural stability, disruption of cytoskeletal elements, inability tomaintain membrane potential, arrest of cell cycle, inability to generateenergy, etc. Thus, host cells containing target polynucleotides may berecovered, i.e., separated from remaining cells, by any physical meanssuch as aspiration, washing, filtration, centrifugation, cell sorting,fluorescence activated cell sorting (FACS), etc.

[0228] For example, host cells containing polynucleotides encodingimmunoglobulin subunit polypeptides may lyse and thereby releaserecombinant virus particles, preferably poxvirus particles even morepreferably vaccinia virus particles into the culture media or may becomenonadherent and therefore lift away from the solid support. Thus, in apreferred embodiment, released recombinant viruses and/or nonadherentcells are separated from adherent cells by aspiration or washing.

[0229] Where the host cells are an early B cell lymphoma cell line, thecells may be attached to a solid substrate through interaction with a Bcell-specific antibody which has been bound to the substrate. Suitable Bcell-specific antibodies include, but are not limited to an anti-CD 19antibody and an anti-CD 20 antibody.

[0230] In other preferred embodiments, antigen-induced cell death iseffected directly or indirectly by employing a host cell transfectedwith a construct in which a foreign polynucleotide, the expression ofwhich indirectly results in cell death, is operably associated with atranscriptional regulatory region which is induced upon cross-linking ofsurface immunoglobulin molecules.

[0231] By a “transcriptional regulatory region induced uponcross-linking of surface immunoglobulin molecules” is meant a region,for example, a host cell promoter, which normally regulates a gene thatis upregulated in the host cell upon cross linking of surface-expressedimmunoglobulin molecules. A preferred example of such a transcriptionalregulatory region is the BAX promoter, which is upregulated in early Bcell lymphoma cells upon cross linking of surface immunoglobulinmolecules.

[0232] In one embodiment, illustrated in FIG. 2A and FIG. 2B, a methodis provided to induce cell death upon expression of a foreignpolynucleotide encoding a cytotoxic T cell (CTL) epitope. The foreignpolynucleotide encoding the CTL epitope is placed in operableassociation with a transcriptional regulatory region which is inducedupon cross-linking of surface immunoglobulin molecules. Uponantigen-induced cross-linking of immunoglobulin molecules of the surfaceof host cells, the CTL epitope is expressed on the surface of the hostcell in the context of a defined MLC molecule, also expressed on thesurface of the host cell. The cells are contacted with epitope-specificCTLs which recognize the CTL epitope in the context of the defined MHCmolecule, and the cells expressing the CTL epitope rapidly undergo alytic event. Methods of selecting and recovering host cells expressingspecific CTL epitopes are further disclosed in Zauderer, PCT PublicationNo. WO 00/028016.

[0233] Selection of the host cells is accomplished through recoveringthose cells, or the contents thereof, which have succumbed to cell deathand/or have undergone a lytic event. For example, if host cells arechosen which grow attached to a solid support, those host cells whichsuccumb to cell death and/or undergo a lytic event will be released fromthe support and can be recovered in the cell supernatant. Alternativelyvirus particles released from host cells which have succumbed to celldeath and/or undergone a lytic event may be recovered from the cellsupernatant.

[0234] According to this embodiment, the MHC molecule expressed on thesurface of the host cells may be either a class I MHC molecule or aclass II MHC molecule. In a particularly preferred embodiment, the MHCmolecule expressed on the host cells is an H-2K^(d) molecule, and theCTL epitope which is expressed upon antigen-induced cross linking is thepeptide GYKAGMIHI, designated herein as SEQ ID NO:23.

[0235] In utilizing this method, any host cell which is capable ofexpressing immunoglobulin molecules, or antigen-specific fragmentsthereof, on its surface maybe used. Suitable host cells includeimmunoglobulin-negative plasmacytoma cell lines. Examples of such celllines include, but are not limited to, an NS 1 cell line, an Sp2/0 cellline, and a P3 cell line. Other suitable cell lines will be apparent tothose of ordinary skill in the art.

[0236] In another preferred embodiment, also illustrated in FIG. 2A andFIG. 2B, a method is provided wherein cell death is induced indirectlyby employing a host cell transfected with a construct in which the aheterologous polynucleotide comprising a “suicide” gene is operablyassociated with a transcriptional regulatory region which is inducedupon cross-linking of surface immunoglobulin molecules. By “suicidegene” is meant a nucleic acid molecule which causes cell death whenexpressed. Polynucleotides useful as suicide genes include many celldeath-inducing sequences which are known in the art. Preferred suicidegenes are those which encode toxins such as Pseudomonas exotoxin Achain, diphtheria A chain, ricin A chain, abrin A chain, modeccin Achain, and alpha-sarcin. A preferred suicide gene encodes the diphtheriaA toxin subunit. Upon antigen-induced cross-linking of immunoglobulinmolecules of the surface of host cells, the promoter of the apoptosisinduced gene is induced, thereby allowing expression of the suicidegene, and thereby promoting cell death.

[0237] In utilizing this method, any host cell may be used which iscapable of expressing immunoglobulin molecules, or antigen-specificfragments thereof, on its surface, and in which a transcriptionalregulatory region can be identified by expression profiling, which isinduced upon cross-linking of surface immunoglobulin molecules. Suitablehost cells include early B cell lymphoma cell lines andimmunoglobulin-negative plasmacytoma cell lines. Examples of such celllines include, but are not limited to, a CH33 cell line, a CH 31 cellline, a WEHI-231 cell line, an NS1 cell line, an Sp2/0 cell line, and aP3 cell line. Other suitable cell lines will be apparent to those ofordinary skill in the art.

[0238] Where the host cells are an Ig-negative plasmacytoma cell line,the cells may be attached to a solid substrate through interaction witha plasmacytoma-specific antibody which has been bound to the substrate.Suitable plasmacytoma-specific antibodies include, but are not limitedto an anti-CD38 antibody (Yi, Q., et al., Blood 90:1960-1967 (1997)), ananti-CD31 antibody (Medina, F., et al., Cytometry 39:231-234 (2000)), ananti-CD20 antibody (Haghighi, B., et al., Am. J. Hematol. 59:302-308(1998)), and an anti-CD 10 antibody (Dunphy, C. H., Acta. Cytol.40:358-362 (1996)).

[0239] Direct and indirect antigen-induced cell death methods asdescribed herein may also be combined. For example, in those embodimentswhere the host cell is an early B cell lymphoma, and antigencross-linking directly induces apoptosis, antigen-induced cell death maybe accelerated by transfecting the early B cell lymphoma host cell witha construct in which the a polynucleotide encoding a foreign cytotoxic Tcell epitope is operably associated with a transcriptional regulatoryregion which is induced upon cross-linking of surface immunoglobulinmolecules. Upon contacting antigen cross-linked cells with specificcytotoxic T cells as described, cell death is accelerated. Similarly, inthose embodiments where the host cell is an early B cell lymphoma, andantigen cross-linking directly effects apoptosis as described above,antigen-induced cell death may be accelerated by transfecting the earlyB cell lymphoma host cell with a construct in which a suicide gene isoperably associated with a transcriptional regulatory region which isinduced upon cross-linking of surface immunoglobulin molecules.

[0240] Immunoglobulin heavy chains can be modified so that a specificantigen will induce a readily detectable signal in cells in which thereceptor is crosslinked by specific antigen. A preferred embodiment isto use an apoptosis induction system to select for cell killing as aconsequence of expression of an antigen-specific receptor. An example ofan apoptosis induction system involves the human FAS (CD95, APO-1)receptor, which is a member of the tumor necrosis-nerve growth factorreceptor superfamily recognized for its role in regulating apoptosisthrough recruitment and assembly of a death-inducing signaling complexthat activates a cascade of proteolytic caspases. Several reports havedescribed a FAS-based inducible cell death system whereby apoptosiscould be induced through chimeric proteins containing the cytoplasmic“death domain” of FAS coupled to various receptors allowing forinduction of apoptosis through a variety of cell modulators.Ishiwatari-Hayasaka et al. have successfully used the extracellulardomain of mouse CD44 with human FAS to induce apoptosis uponcross-linking with polyvalent anti-CD44 antibodies (Ishiwatari-HayasakaH. et al. J Immunol 163:1258-64 (1999)). In addition, Takahashi et al.have demonstrated that a chimeric human G-CSFR/FAS (extracellular/cytoplasmic) protein is capable of inducing apoptosis uponcross-linking with anti-G-CSFR antibodies (Takahashi T. et al. J BiolChem 271:17555-60 (1996)). These authors also demonstrate that thechimeric protein is incapable of inducing apoptosis as a dimer. Thecomplex must be in at least a trimeric form.

[0241] In a preferred embodiment, a chimeric gene is constructed inwhich the transmembrane domain and cytoplasmic death domain of FAS isfused to the carboxyl terminus of the CH1 domain of the human IgM heavychain (CH1-Fas, FIG. 13(a)). Diverse VH genes are inserted into thisconstruct as described herein to create a library of VH-CH1-Fasrecombinant vaccinia virus. Membrane receptors with VH-CH1-Fas areassembled in host cells which are infected with the VH-CH1-Fasconstructs and which are transfected with DNA encoding diverseimmunoglobulin light chains or which are infected with psoralin treatedrecombinant vaccinia virus encoding diverse immunoglobulin light chains.Those cells that express a combination of heavy and light chain variableregion genes with a desired specificity will have some of their membranereceptors crosslinked in the presence of the specific immobilizedantigen of interest. Apoptosis will be induced as a result of formationof functional complexes of VHCH1/FAS oligomers. Trimer formation canoccur through crosslinking with polyvalent antigens or throughimmobilization of more than one antigen to tissue culture plates orbeads.

[0242] In an alternative embodiment, the VH library is expressed infusion proteins in which a polypeptide comprising the transmembranedomain and cytoplasmic death domain of FAS is fused to the carboxylterminus of the IgM heavy chain CH4 domain (FIG. 13 (b)). In yet anotherembodiment, the cytoplasmic death domain of FAS is fused to the carboxylterminus of the IgM heavy chain transmembrane domain following the CH4domain (FIG. 13(c)).

[0243] The latter two embodiments (FIG. 13(b and c)) result in synthesisof an already dimeric Fas death domain which facilitates formation oftrimeric complexes required for induction of the apoptotic signal andthereby increases the number of antigen-specific receptors selected. Useof the monomeric construct (FIG. 13(a)), however, results in selectionof fewer but higher affinity antigen receptors, and also reduces thebackground of non-antigen specific cell death. The two receptors withdimeric Fas domains differ in terms of whether the transmembrane regionencoded in the fusion protein is Fas-derived or IgM-derived. AnIgM-derived transmembrane region may function more efficiently formembrane receptor expression in cells of the B lymphocyte lineage. Anadvantage of this embodiment, however, is that it is not limited to Bcells. In particular, the monomeric Fas construct is synthesized andexpressed as a membrane receptor in a wide variety of cell typesincluding epithelial cell lines, Hela cells and BSC-1 cells in whichhigh titers of vaccinia virus can be generated.

[0244] In another embodiment, a screening method is provided to recoverpolynucleotides encoding immunoglobulin molecules, or antigen-specificfragments thereof, based on antigen-induced cell signaling. According tothis method, host cells are transfected with an easily detected reporterconstruct, for example luciferase, operably associated with atranscriptional regulatory region which is upregulated as a result ofsurface immunoglobulin crosslinking. Pools of host cells expressingimmunoglobulins or fragments thereof on their surface are contacted withantigen, and upon cross linking, the signal is detected in that pool.Referring to the first step in the immunoglobulin identification methodas described above, the signaling method may be carried out as follows.The first library of polynucleotides encoding immunoglobulin subunitpolypeptides is divided into a plurality of pools, e.g., about 2, 5, 10,25, 15, 75, 100, or more pools, each pool containing about 10, 100, 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different polynucleotides encodingimmunoglobulin subunit polypeptides with different variable regions.Preferred pools initially contain about 10³ polynucleotides each. Eachpool is expanded and a replicate aliquot is set aside for laterrecovery. Where the pools of polynucleotides are constructed in virusvectors, preferably poxvirus vectors, and even more preferably vacciniavirus vectors, the pools are prepared, e.g., by diluting a high-titerstock of the virus library and using the portions to infectmicrocultures of tissue culture cells at a low MOI, e.g., MOI<0.1.Typically a greater than 1,000 fold expansion in the viral titer isobtained after 48 hrs infection. Expanding viral titers in multipleindividual pools mitigates the risk that a subset of recombinants willbe lost due to relatively rapid growth of a competing subset.

[0245] The virus pools are then used to infect pools of host cells equalto the number of virus pools prepared. These host cells have beenengineered to express a reporter molecule as a result of surfaceimmunoglobulin crosslinking. The number of host cells infected with eachpool depends on the number of polynucleotides contained in the pool, andthe MOI desired. The second library of polynucleotides is alsointroduced into the host cell pools, and expression of immunoglobulinmolecules or fragments thereof on the surface of the host cells ispermitted.

[0246] The host cell pools are then contacted with a desired antigenunder conditions wherein host cells expressing antigen-specificimmunoglobulin molecules on their surface express the detectablereporter molecule upon cross-linking of said immunoglobulin molecules,and the various pools of host cells are screened for expression of thereporter molecule. Those pools of host cells in which reporterexpression is detected are harvested, and the polynucleotides of thefirst library contained therein are recovered from the aliquotpreviously set aside following initial expansion of that pool ofpolynucleotides.

[0247] To further enrich for polynucleotides of the first library whichencode antigen-specific immunoglobulin subunit polypeptides, thepolynucleotides recovered above are divided into a plurality ofsub-pools. The sub-pools are set to contain fewer different members thanthe pools utilized above. For example, if each of the first poolscontained 10³ different polynucleotides, the sub-pools are set up so asto contain, on average, about 10 or 100 different polynucleotides each.The sub-pools are introduced into host cells with the second library asabove, and expression of immunoglobulin molecules, or fragments thereof,on the membrane surface of the host cells is permitted. The host cellsare then contacted with antigen as above, and those sub-pools of hostcells in which expression of the reporter molecule is detected areidentified, and the polynucleotides of the first library containedtherein are recovered from the replicate pools previously set aside asdescribed above. It will be appreciated by those of ordinary skill inthe art that this process may be repeated one or more additional timesin order to adequately enrich for polynucleotides encodingantigen-specific immunoglobulin subunit polypeptides.

[0248] Upon further selection and enrichment steps for polynucleotidesof the first library, and isolation or those polynucleotides, a similarprocess is carried out to recover polynucleotides of the second librarywhich, as part of an immunoglobulin molecule, or antigen-specificfragment thereof, bind the desired specific antigen.

[0249] Any suitable reporter molecule may be used in this method, thechoice depending upon the host cells used, the detection instrumentsavailable, and the ease of detection desired. Suitable reportermolecules include, but are not limited to luciferase, green fluorescentprotein, and beta-galactosidase.

[0250] Any host cell capable of expressing immunoglobulin molecules onits surface may be used in this method. Preferred host cells includeimmunoglobulin-negative plasmacytoma cells, e.g., NS1 cells, Sp2/0cells, or P3 cells, and early B-cell lymphoma cells.

[0251] Similar to the cell death methods described above, kineticconsiderations dictate that expression of the reporter construct takeplace prior to the induction of CPE. Nonetheless, it is preferred thatexpression of a detectable reporter molecule in response toantigen-induced cross-linking of immunoglobulin molecules on the surfaceof the host cells occurs within a period between about 1 hour to about 4days after contacting the host cells with antigen, so as to precedeinduction of CPE. More preferably, reporter molecule expression occurswithin about 1 hour about 2 hours, about 3 hours about 4 hours, about 5hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about10 hours, about 11 hours, about 12 hours, about 14 hours, about 16hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours,about 28 hours, about 32 hours, about 36 hours, about 40 hours, about 44hours, or about 48 hours after contacting the host cells with antigen.Even more preferably reporter molecule expression occurs within about 12hours of contacting the host cells with antigen.

[0252] By a “transcriptional regulatory region induced uponcross-linking of surface immunoglobulin molecules” is meant a region,for example, a host cell promoter, which normally regulates a gene thatis upregulated in the host cell upon cross linking of surface-expressedimmunoglobulin molecules. A preferred example of such a transcriptionalregulatory region is the BAX promoter, which is upregulated in early Bcell lymphoma cells upon cross linking of surface immunoglobulinmolecules.

[0253] In yet another embodiment, a selection or screening method isprovided to select polynucleotides encoding immunoglobulin molecules, orantigen-specific fragments thereof, based on antigen-specific binding.This embodiment is illustrated in FIG. 5. According to this method, hostcells which express antigen-specific immunoglobulin molecules, orfragments thereof on their surface are recovered based solely on thedetection of antigen binding. Antigen binding may be utilized as aselection method, i.e., where host cells expressing antigen-specificimmunoglobulin molecules are directly selected by virtue of bindingantigen, by methods similar to those described for selection based oncell death as described above. For example, if an antigen is bound to asolid substrate, host cells in suspension which bind the antigen may berecovered by binding, through the antigen, to the solid substrate.Alternatively, antigen binding may be used as a screening process, i.e.,where pools of host cells are screened for detectable antigen binding bymethods similar to that described above for antigen-induced cellsignaling. For example, pools of host cells expressing immunoglobulinsor fragments thereof on their surface are contacted with antigen, andantigen binding in a given pool is detected through an immunoassay, forexample, through detection of an enzyme-antibody conjugate which bindsto the antigen.

[0254] Referring to the first step in the immunoglobulin identificationmethods as described above, selection via the antigen-specific bindingmethod may be carried out as follows. A host cell is selected forinfection and/or transfection that is capable of high level expressionof immunoglobulin molecules on its surface. Preferably, the host cellgrows in suspension. Following infection with the first and secondpolynucleotide libraries as described above, synthesis and assembly ofantibody molecules is allowed to proceed. The host cells are thentransferred into microtiter wells which have antigen bound to theirsurface. Host cells which bind antigen thereby become attached to thesurface of the well, and those cells that remain unbound are removed bygentle washing. Alternatively, host cells which bind antigen may berecovered, for example, by fluorescence-activated cell sorting (FACS).FACS, also called flow cytometry, is used to sort individual cells onthe basis of optical properties, including fluorescence. It is usefulfor screening large populations of cells in a relatively short period oftime. Finally the host cells which bound to the antigen are recovered,thereby enriching for polynucleotides of the first library which encodea first immunoglobulin subunit polypeptide which, as part of animmunoglobulin molecule, or antigen-specific fragment thereof,specifically binds the antigen of interest.

[0255] Upon further selection and enrichment steps for polynucleotidesof the first library, and isolation or those polynucleotides, a similarprocess is carried out to recover polynucleotides of the second librarywhich, as part of an immunoglobulin molecule, or antigen-specificfragment thereof, bind the desired specific antigen.

[0256] Any host cell capable of expressing immunoglobulin molecules onits surface may be used in this selection method. Preferred host cellsinclude immunoglobulin-negative plasmacytoma cells, e.g., NS 1 cells,Sp2/0 cells, or P3 cells, and early B-cell lymphoma cells. It ispreferred that the cells are capable of growth in suspension.

[0257] Referring to the first step in the immunoglobulin identificationmethods as described above, screening via the antigen-specific bindingmethod may be carried out as follows. The first library ofpolynucleotides, constructed in a virus vector encoding immunoglobulinsubunit polypeptides, is divided into a plurality of pools by the methoddescribed above. The virus pools are then used to infect pools of hostcells equal to the number of virus pools prepared. In this screeningmethod, it is preferred that the host cells are adherent to a solidsubstrate. The second library of polynucleotides is also introduced intothe host cell pools, and expression of immunoglobulin molecules orfragments thereof on the surface of the host cells is permitted.

[0258] The host cell pools are then contacted with a desired antigen.Following incubation with the antigen, excess unbound antigen is washedaway. Finally the pools of cells are screened for antigen binding.Antigen binding may be detected by a variety of methods. For example, anantigen may be conjugated to an enzyme. Following the removal of unboundantigen, substrate is added, and enzyme reaction products are detected.This method may be enhanced by use of a secondary antibody conjugate, ora streptavidin/biotin system. Such screening methods are well known tothose of ordinary skill in the art, and are readily available in kitform from standard vendors. Also, if the antigen is bound to microscopicparticles, for example, gold beads, binding of the antigen to the hostcells may be detected microscopically. As with the cell signalingmethods described above, those pools of host cells in which antigenbinding is detected are harvested, and the polynucleotides of the firstlibrary contained therein are recovered. Alternatively, pools of hostcells in which antigen-binding is detected are identified, andpolynucleotides of the first library contained therein are recoveredfrom a replicate aliquot of that pool of polynucleotides set asidefollowing initial expansion of the library.

[0259] To further enrich for polynucleotides of the first library whichencode antigen-specific immunoglobulin subunit polypeptides, thepolynucleotides recovered above are divided into a plurality ofsub-pools. The sub-pools are set to contain fewer different members thanthe pools utilized in the first round. For example, if each of the firstpools contained 10³ different polynucleotides, the sub-pools are set upso as to contain, on average, about 10 or 100 different polynucleotideseach. The sub-pools are introduced into host cells with the secondlibrary as above, and expression of immunoglobulin molecules, orfragments thereof, on the membrane surface of the host cells ispermitted. The host cells are then contacted with antigen as above, andthose sub-pools of host cells in which antigen binding is detected areharvested or simply identified, and the polynucleotides of the firstlibrary contained therein, or in a replicate aliquot, are recovered. Itwill be appreciated by those of ordinary skill in the art that thisprocess may be repeated one or more additional times in order toadequately enrich for polynucleotides encoding antigen-specificimmunoglobulin subunit polypeptide.

[0260] Upon further selection and enrichment steps for polynucleotidesof the first library, and isolation or those polynucleotides, a similarprocess is carried out to recover polynucleotides of the second librarywhich, as part of an immunoglobulin molecule, or antigen-specificfragment thereof, bind the desired specific antigen.

[0261] Any host cell capable of expressing immunoglobulin molecules onits surface may be used in this method. Preferred host cells includeimmunoglobulin-negative plasmacytoma cells, e.g., NS1 cells, Sp2/0cells, or P3 cells, and early B-cell lymphoma cells.

[0262] An antigen of interest may be contacted with host cells by anyconvenient method when practicing the direct and indirectantigen-induced cell death methods as described herein. For example, incertain embodiments, antigen, for example a peptide or a polypeptide, isattached to a solid substrate. As used herein, a “solid support” or a“solid substrate” is any support capable of binding a cell or antigen,which may be in any of various forms, as is known in the art. Well-knownsupports include tissue culture plastic, glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, gabbros, and magnetite. The natureof the carrier can be either soluble to some extent or insoluble for thepurposes of the present invention. The support material may havevirtually any possible structural configuration as long as the coupledmolecule is capable of binding to a cell. Thus, the supportconfiguration may be spherical, as in a bead, or cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports include polystyrene beads. The support configurationmay include a tube, bead, microbead, well, plate, tissue culture plate,petri plate, microplate, microtiter plate, flask, stick, strip, vial,paddle, etc., etc. A solid support may be magnetic or non-magnetic.Those skilled in the art will know many other suitable carriers forbinding cells or antigens, or will be able to readily ascertain thesame.

[0263] Alternatively, an antigen is expressed on the surface of anantigen-expressing presenting cell. As used herein an“antigen-expressing presenting cell” refers to a cell which expresses anantigen of interest on its surface in a manner such that the antigen mayinteract with immunoglobulin molecules attached to the surface of hostcells of the present invention. An preferred antigen-expressingpresenting cell is engineered such that it expresses the antigen ofinterest as a recombinant protein, but the antigen may be a nativeantigen of that cell. Recombinant antigen-expressing presenting cellsmay be constructed by any suitable method using molecular biology andprotein expression techniques well-known to those of ordinary skill inthe art. Typically, a plasmid vector which encodes the antigen ofinterest is transfected into a suitable cell, and the cell is screenedfor expression of the desired polypeptide antigen. Preferred recombinantantigen-expressing presenting cells stably express the antigen ofinterest. A cell of the same type as the antigen-expressing presentingcell except that it has not been engineered to express the antigen ofinterest is referred to herein as an “antigen-free presenting cell.” Anysuitable cell line may be used to prepare antigen-expressing presentingcells. Examples of cell lines include, but are not limited to: monkeykidney CVIline transformed by SV40 (COS-7, ATCC CRL 165 1); humanembryonic kidney line (293, Graham et al. J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamsterovary-cells-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. (USA)77:4216, (1980); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); african greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals N. Y. Acad.Sci 383:44-68 (1982)); NIH/3T3 cells (ATCC CRL-1658); and mouse L cells(ATCC CCL-1). Additional cell lines will become apparent to those ofordinary skill in the art. A wide variety of cell lines are availablefrom the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209.

[0264] As will be appreciated by those of ordinary skill in the art,antigen-expressing presenting cells will comprise manynaturally-occurring antigenic determinants on their surface in additionto the antigen of interest. Certain of the host cells of the presentinvention which express a broad spectrum of different immunoglobulinmolecules, or antigen-specific fragments thereof on their surface wouldbe expected to bind to these additional antigenic determinants.Accordingly, when an antigen-expressing presenting cell is used tocontact host cells of the invention with the antigen of interest, it isnecessary to first deplete the host cell population of those host cellswhich express immunoglobulins reactive for these additional antigenicdeterminants. The present invention provides methods to deplete the hostcell population of host cells expressing immunoglobulin moleculesspecific for naturally-occurring surface antigens of the antigen-freepresenting cell. This is illustrated in FIG. 5. Essentially, thesemethods comprise contacting the host cell population with antigen-freepresenting cells prior to contacting the population of host cells withantigen-expressing presenting cells.

[0265] In one embodiment, this method comprises adsorbing the populationof host cells to antigen-free presenting cells which are bound to asolid substrate. The unbound cells and/or the polynucleotides containedtherein are recovered, and the recovered host cells, or new host cellsinto which the recovered polynucleotides have been introduced, are thencontacted with antigen-expressing presenting cells. In those selectionmethods where pools of host cells are contacted with antigen, the poolsof host cells are adsorbed to antigen-free presenting cells bound to asolid substrate. The unbound cells in the pool and/or thepolynucleotides contained therein, are recovered, and the recovered hostcells, or host cells into which the recovered polynucleotides have beenintroduced, are then contacted with antigen-expressing presenting cells.

[0266] In another embodiment, the method comprises contacting thepopulation of host cells with antigen-free presenting cells underconditions wherein host cells expressing surface immunoglobulinmolecules which react with surface antigens of antigenic determinants onthe antigen-free presenting cells undergo either programmed cell death,e.g., apoptosis, direct or indirect cell death, or cell signaling, i.e.,expression of a reporter molecule, all as described above, uponcross-linking of immunoglobulin molecules on the surface of the hostcells. Those host cells, and more specifically, polynucleotides fromeither the first library or second library, from those host cells whichhave not succumbed to cell death or do not express a reporter molecule,are then recovered. For example, if the host cell population expressingimmunoglobulin molecules is maintained attached to a solid substrate,and those cells which undergo cell death are released from thesubstrate, the contents of the culture fluid are removed and discarded,and the cells which remain attached, and the polynucleotides containedtherein, are recovered.

[0267] As will be appreciated by those of ordinary skill in the art,depleting the host cell population of those host cells which expressimmunoglobulins reactive with determinants carried on the antigen-freepresenting cells may require more than one round of depletion. It isfurther contemplated that successive rounds of depletion may bealternated with successive rounds of enrichment for host cellsexpressing immunoglobulin molecules which specifically bind to theantigen of interest expressed on the antigen-expressing presentingcells.

[0268] In yet another embodiment, a screening method is provided torecover polynucleotides encoding immunoglobulin molecules, orantigen-specific functional fragments thereof, based on a desiredantigen-specific function of the immunoglobulin molecule. According tothis method, pools of host cells are prepared which expressfully-soluble immunoglobulin molecules. Expression is permitted, and theresulting cell medium is tested in various functional assays whichrequire certain desired antigenic specificities. According to thismethod, the “function” being tested may be a standard effector functioncarried out by an immunoglobulin molecule, e.g., virus neutralization,opsonization, ADCC, antagonist/agonist activity, histamine release,hemagglutination, or hemagglutination inhibition. Alternatively, the“function” may simply refer to binding an antigen.

[0269] In a related embodiment, a screening method is provided to selectimmunoglobulin molecules of a known antigenic specificity, but withaltered effector functions. According to these embodiments, libraries ofimmunoglobulin subunit polypeptides with a known antigenic specificity,but with alterations in constant domain regions known to be involved ina given effector function, are constructed. According to this method,pools of host cells are prepared which express fully-solubleimmunoglobulin molecules. Expression is permitted, and the resultingcell medium is tested in various functional assays for improved orsuppressed activity. According to this method, the “function” beingtested may be a standard effector function carried out by animmunoglobulin molecule, e.g., virus neutralization, opsonization,complement binding, ADCC, antagonist/agonist activity, histaminerelease, hemagglutination, or hemagglutination inhibition.

[0270] Referring to the first step in the immunoglobulin identificationmethod as described above, the screening for effector function may becarried out as follows. The first library of polynucleotides encodingfully secreted immunoglobulin subunit polypeptides is divided into aplurality of pools, as described above, each pool containing about 10,100, 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, or 10⁹ different polynucleotidesencoding fully-secreted immunoglobulin subunit polypeptides withdifferent variable regions. Preferred pools initially contain about 10³polynucleotides each. Each pool is expanded and a replicate aliquot isset aside for later recovery. Where the pools of polynucleotides areconstructed in virus vectors, preferably poxvirus vectors, and even morepreferably vaccinia virus vectors, the pools are prepared, e.g., bydiluting a high-titer stock of the virus library and using the portionsto infect microcultures of tissue culture cells at a low MOI, e.g.,MOI<0.1. Typically a greater than 1,000 fold expansion in the viraltiter is obtained after 48 hrs infection. Expanding viral titers inmultiple individual pools mitigates the risk that a subset ofrecombinants will be lost due to relatively rapid growth of a competingsubset.

[0271] The virus pools are then used to infect pools of host cells equalto the number of virus pools prepared. The number of host cells infectedwith each pool depends on the number of polynucleotides contained in thepool, and the MOI desired. Virtually any host cell which is permissivefor infection with the virus vector used, and which is capable ofexpressing fully-secreted immunoglobulin molecules may be used in thismethod. Preferred host cells include immunoglobulin-negativeplasmacytoma cells, e.g., NS1 cells, Sp2/0 cells, or P3 cells, and earlyB-cell lymphoma cells. The cells may be cultured in suspension orattached to a solid surface. The second library of polynucleotides isalso introduced into the host cell pools, and expression of fullysecreted immunoglobulin molecules or fragments thereof is permitted.

[0272] The conditioned medium in which the host cell pools were culturedis then recovered and tested in a standardized functional assay foreffector function in response to a specific target antigen.

[0273] Any suitable functional assay may be used in this method. Forexample, the harvested cell supernatants may be tested in a virusneutralization assay to detect immunoglobulin molecules with the abilityto neutralize a target virus, for example, HIV. Alternatively, theharvested cell supernatants may be tested for the ability to block orfacilitate, i.e., act as an antagonist or an agonist of, a targetcellular function, for example, apoptosis. Exemplary suitable functionalassays are described in the Examples, infra. As used herein, a“functional assay” also included simple detection of antigen binding,for example, through use of a standard ELISA assay, which is well knownto those of ordinary skill in the art.

[0274] Where the conditioned medium in which a given host cell pool wasgrown exerts the desired function, the polynucleotides of the firstlibrary contained in host cells of that pool are recovered from thealiquot previously set aside following initial expansion of that pool ofpolynucleotide.

[0275] To further enrich for polynucleotides of the first library whichencode antigen-specific immunoglobulin subunit polypeptides, thepolynucleotides recovered above are divided into a plurality ofsub-pools. The sub-pools are set to contain fewer different members thanthe pools utilized above. For example, if each of the first poolscontained 10³ different polynucleotides, the sub-pools are set up so asto contain, on average, about 10 or 100 different polynucleotides each.The sub-pools are introduced into host cells with the second library asabove, and expression of fully secreted immunoglobulin molecules, orfragments thereof, is permitted. The conditioned medium in which thehost cell pools are cultured is is recovered and tested in astandardized functional assay for effector function in response to aspecific target antigen as described above, conditioned media sampleswhich possess the desired functional characteristic are identified, andthe polynucleotides of the first library contained in host cells of thatsub-pool are recovered from the aliquot previously set aside asdescribed above. It will be appreciated by those of ordinary skill inthe art that this process may be repeated one or more additional timesin order to adequately enrich for polynucleotides encodingantigen-specific immunoglobulin subunit polypeptides.

[0276] Upon further selection and enrichment steps for polynucleotidesof the first library, and isolation of those polynucleotides, a similarprocess is carried out to recover polynucleotides of the second librarywhich, as part of an fully secreted immunoglobulin molecule, or fragmentthereof, exhibits the desired antigen-specific function.

[0277] Kits. The present invention further provides a kit for theselection of antigen-specific recombinant immunoglobulins expressed in aeukaryotic host cell. The kit comprises one or more containers filledwith one or more of the ingredients required to carry out the methodsdescribed herein. In one embodiment, the kit comprises: (a) a firstlibrary of polynucleotides encoding, through operable association with atranscriptional control region, a plurality of first immunoglobulinsubunit polypeptides, where each first immunoglobulin subunitpolypeptide comprises (i) a first immunoglobulin constant regionselected from the group consisting of a heavy chain constant region anda light chain constant region, (ii) an immunoglobulin variable regioncorresponding to said first constant region, and (iii) a signal peptidecapable of directing cell surface expression or secretion of said firstimmunoglobulin subunit polypeptide, wherein said first library isconstructed in a eukaryotic virus vector; (b) a second library ofpolynucleotides encoding, through operable association with atranscriptional control region, a plurality of second immunoglobulinsubunit polypeptides, where each comprises: (i) a second immunoglobulinconstant region selected from the group consisting of a heavy chainconstant region and a light chain constant region, wherein said secondimmunoglobulin constant region is not the same as the firstimmunoglobulin constant region, (ii) an immunoglobulin variable regioncorresponding to said second constant region, and (iii) a signal peptidecapable of directing cell surface expression or secretion of said secondimmunoglobulin subunit polypeptide, where the second immunoglobulinsubunit polypeptide is capable of combining with the firstimmunoglobulin subunit polypeptide to form a surface immunoglobulinmolecule, or antigen-specific fragment thereof, attached to the membraneof a host cell, and where the second library is constructed in aeukaryotic virus vector; and (c) a population of host cells capable ofexpressing said immunoglobulin molecules. In this kit, the first andsecond libraries are provided both as infectious virus particles and asinactivated virus particles, where the inactivated virus particles arecapable of infecting the host cells and allowing expression of thepolynucleotides contained therein, but the inactivated viruses do notundergo virus replication. In addition, the host cells provided with thekit are capable of expressing an antigen-specific immunoglobulinmolecule which can be selected through interaction with an antigen. Useof the kit is in accordance to the methods described herein. In certainembodiments the kit will include control antigens and reagents tostandardize the validate the selection of particular antigens ofinterest.

[0278] Isolated immunoglobulins. The present invention further providesan isolated antigen-specific immunoglobulin, or fragment thereof,produced by any of the methods disclosed herein. Such isolatedimmunoglobulins may be useful as diagnostic or therapeutic reagents.Further provided is a composition comprising an isolated immunoglobulinof the present invention, and a pharmaceutically acceptable carrier.

[0279] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See, for example,Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed.,Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: ALaboratory Manual, Sambrook et al., ed., Cold Springs Harbor Laboratory,New York (1992), DNA Cloning, Volumes I and II (D. N. Glover ed., 1985);Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S.Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J.Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise,Methods In Enzymology (Academic Press, Inc., New York); Gene TransferVectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987,Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155(Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987); Handbook OfExperimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell,eds., 1986); Manipulating the Mouse Embryo, (Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York, 1986); and in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989).

[0280] General principles of antibody engineering are set forth inAntibody Engineering, 2nd edition, C. A. K. Borrebaeck, Ed., OxfordUniv. Press (1995). General principles of protein engineering are setforth in Protein Engineering, A Practical Approach, Rickwood, D., etal., Eds., IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). Generalprinciples of antibodies and antibody-hapten binding are set forth in:Nisonoff, A., Molecular Immunology, 2nd ed., Sinauer Associates,Sunderland, Mass. (1984); and Steward, M. W., Antibodies, TheirStructure and Function, Chapman and Hall, New York, N.Y. (1984).Additionally, standard methods in immunology known in the art and notspecifically described are generally followed as in Current Protocols inImmunology, John Wiley & Sons, New York; Stites et al. (eds), Basic andClinical—Immunology (8th ed.), Appleton & Lange, Norwalk, Conn. (1994)and Mishell and Shiigi (eds), Selected Methods in Cellular Immunology,W. H. Freeman and Co., New York (1980).

[0281] Standard reference works setting forth general principles ofimmunology include CurrentProtocols in Immunology, John Wiley & Sons,New York; Klein, J., Immunology: The Science of Self-NonselfDiscrimination, John Wiley & Sons, New York (1982); Kennett, R., et al.,eds., Monoclonal Antibodies, Hybridoma: A New Dimension in BiologicalAnalyses, Plenum Press, New York (1980); Campbell, A., “MonoclonalAntibody Technology” in Burden, R., et al., eds., Laboratory Techniquesin Biochemistry and Molecular Biology, Vol. 13, Elsevere, Amsterdam(1984).

EXAMPLES Example 1 Construction of Human Immunoglobulin Libraries ofDiverse Specificity

[0282] Libraries of polynucleotides encoding diverse immunoglobulinsubunit polypeptides are produced as follows. Genes for human VH(variable region of heavy chain), VK (variable region of kappa lightchain) and VL (variable region of lambda light chains) are amplified byPCR. For each of the three variable gene families, both a recombinantplasmid library and a vaccinia virus library is constructed. Thevariable region genes are inserted into a p7.5/tk-basedtransfer/expression plasmid between immunoglobulin leader and constantregion sequences of the corresponding heavy chain or light chain. Thisplasmid is employed to generate the corresponding vaccinia virusrecombinants by trimolecular recombination and can also be used directlyfor high level expression of immunoglobulin chains followingtransfection into vaccinia virus infected cells. Lymphoma cells arefirst infected with the vaccinia heavy chain library, followed bytransient transfection with a plasmid light chain library. Theco-expression of IgM and light chain results in the assembly and surfaceexpression of antibody molecules.

[0283] 1.1 pVHE. An expression vector comprising the human μ membraneimmunoglobulin constant region, designated herein as pVHE is constructedas follows. The strategy is depicted in FIG. 3. A cDNA coding for themembrane-bound human IgM heavy chain is isolated from bone marrow RNAusing SMART™ RACE cDNA Amplification Kit available from Clontech, PaloAlto, Calif.. The PCR is carried out using the 5′ primer (huCμ5B)5′-ATTAGGATCC GGTCACCGTC TCCTCAGGG-3′ (SEQ ID NO:24), and 3′ primer(huCu3S) 5′-ATTAGTCGAC TCATTTCACC TTGAACAAGGTGAC-3′ (SEQ ID NO:25). ThePCR product then is inserted into the pBluescript II/KS at BamHI andSalI sites for site-directed mutagenesis to eliminate two BstEII siteslocated in the CH2 and CH4 domains. Nucleotide substitutions areselected that do not alter the amino acids encoded at these sites.

[0284] Plasmid p7 .5/tk, produced as described in Zauderer, PCTPublication No. WO 00/028016, and in Example 5, infra, is converted intopVHE by the following method. The multiple cloning site (MCS) of p7.5/tkis replaced with a cassette containing the following restriction sites:NotI-NcoI-BssHII-BstEII-SalI to generate p7.5/tk2. This cassette, havingthe sequence 5′-GCGGCCGCAA ACCATGGAAA GCGCGCATAT GGTCACCAAAAGTCGAC-3′,is referred to herein as SEQ ID NO:26. A cassette encodingthe signal peptide sequence corresponding to amino acids −19 to −3 ofthe IgM heavy chain is cloned into p7.5/tk2 between the NcoI and BssHIIsites to produce p7.5/tk2L. The BstEII-mutagenized IgM heavy chain,produced as described above, is then cloned into p7.5/tk2L between theBstEII and SalI sites to generate pVHE. Heavy chain variable region (VH)cassettes comprising nucleotides encoding amino acids −4 to 110,produced by PCR as described below, are then cloned between the BssHIIand BstEII sites of pVHE to generate a library of polynucleotidesencoding membrane-bound heavy chains. Because of the overlap between theμ heavy chain sequence and the restriction enzyme sites selected, thisresults in expression of contiguous membrane-bound heavy chainimmunoglobulin subunit polypeptides in the correct translational readingframe.

[0285] 1.2 pVHEs. An expression vector comprising the human μ secretoryimmunoglobulin constant region, designated herein as pVHEs isconstructed as follows. The strategy is depicted in FIG. 8. A cDNAcoding for the secretory human IgM heavy chain is isolated from bonemarrow RNA using SMART™ RACE cDNA Amplification Kit. The upstream primerhuCμ5B contains an appended BaamHI and a BstEII site at the 5′ end,followed by amino acids 111-113 of VH and the first amino acid of CμH1.The downstream primer shuCμ3S contains the last 6 amino acids of thesecreted Cμ, followed by a stop codon and a SalI site. These primershave the following sequences: huCμ5B: 5′-ATTAGGATCC GGTCACCGTCTCCTCAGGG-3′ (SEQ ID NO:27); and shuCμ3S: 5′-ATTAGTCGAC TCAGTAGCAGGTGCCAGCTG T-3′ (SEQ ID NO:28).

[0286] The PCR product then is inserted into the pBluescript II/KS atBamHI and SalI sites for site-directed mutagenesis to eliminate twoBstEll sites located in the CH2 and CH4 domains. Nucleotidesubstitutions are selected that do not alter the amino acids encoded atthese sites.

[0287] Plasmid p7.5/tk2L, produced as in section 1.1, is converted intopVHEs by the following method. The BstEII-mutagenized secretory IgMheavy chain, produced as described above, is then cloned into p7.5/tk2Lbetween the BstII and SalI sites to generate pVHEs. Heavy chain variableregion (VH) cassettes comprising nucleotides encoding amino acids −4 to110, produced by PCR as described below, are then cloned between theBssHII and BstEII sites of pVHEs to generate a library ofpolynucleotides encoding secreted heavy chains. Because of the overlapbetween the μ heavy chain sequence and the restriction enzyme sitesselected, this results in expression of contiguous secretory heavy chainimmunoglobulin subunit polypeptides in the correct translational readingframe.

[0288] 1.3 pVKE and pVLE. Expression vectors comprising the human κ andλ immunoglobulin light chain constant regions, designated herein as pVKEand pVLE, are constructed as follows. The strategy is depicted in FIG.4.

[0289] (a) Plasmid p7.5/tk is converted into pVKE by the followingmethod. The two XhoI sites and two HindIII sites of p7.5/tk are removedby fill-in ligation, the 3 ApaLI sites (one at the backbone, one atColE1 ori, and the other at Amp) are removed by standard methods, andthe multiple cloning site (MCS) of p7.5/tk is replaced with a cassettecontaining the following restriction sites:NotI-NcoI-ApaLI-XhoI-HindIII-SalI to generate p7.5/tk3. This cassette,having the sequence 5′-GCGGCCGCCC ATGGATACGT GCACTTGACT CGAGAAGCTTAGTAGTCGAC-3′, is referred to herein as SEQ ID NO:29. A cassetteencoding the signal peptide sequence corresponding to amino acids −19 to−2 of the kappa light chain is cloned into p7.5/tk3 between the NcoI andApaLI sites to generate p7.5/tk3L. A cDNA coding for the Cκ region isisolated from bone marrow RNA using SMART™ RACE cDNA Amplification Kitas described above, with primers to include an XhoI site at the 5′ endof the region encoding amino acids 104-107+Ck, a stop codon, and a SalIsite at its 3′ end. These primers have the following sequences: huCκ5:5′-CAGGACTCGA GATCAAACGA ACTGTGGCTG −3′ (SEQ ID NO:30); huCκ3:5′-AATATGTCGA CCTAACACTC TCCCCTGTTG AAGCTCTTT-3′ (SEQ ID NO:31); andhuCκ3: 5′-AATATGTCGA CCTAACACTC TCCCCTGTTG AAGCTCTT-3′ (SEQ ID NO:32).The Cκ cassette is then cloned into p7.5/tk3L between the XhoI and SalIsites to generate pVKE. Kappa light chain variable region cassettes (VK)comprising nucleotides encoding amino acids −3 to 105, produced by PCRas described below, are then cloned into pVKE between the ApaLI and XhoIsites. Because of the overlap between the κ light chain sequence and therestriction enzyme sites selected, this results in expression ofcontiguous κ light chain immunoglobulin subunit polypeptides in thecorrect translational reading frame.

[0290] (b) Plasmid p7.5/tk3L is converted into pVLE by the followingmethod. A cDNA coding for the Cκ region is isolated from bone marrow RNAusing SMART™ RACE cDNA Amplification Kit as described above, withprimers to include a HindIII site and the region encoding amino acids105 to 107 of V_(λ) at its 5′ end and a stop codon and a SalI site atits 3′ end. These primers have the following sequences: huCλ5:5′-ATTTAAGCTT ACCGTCCTAC GAACTGTGGC TGCACCATCT −3′ (SEQ ID NO:33); andhuCλ3 (SEQ ID NO:31). The CK cassette is then cloned into p7.5/tk3Lbetween the HindIII and SalI sites to generate pVLE. Lambda light chainvariable region cassettes (VL) comprising nucleotides encoding aminoacids −3 to 104, produced by PCR as described below, are then clonedinto pVLE between the ApaLI and HindIII sites. Because of the overlapbetween the λ light chain sequence and the restriction enzyme sitesselected, this results in expression of contiguous λ light chainimmunoglobulin subunit polypeptides in the correct translational readingframe.

[0291] 1.4 Variable Regions. Heavy chain, kappa light chain, and lambdalight chain variable regions are isolated by PCR for cloning in theexpression vectors produced as described above, by the following method.RNA isolated from normal human bone marrow pooled from multiple donors(available from Clontech) is used for cDNA synthesis. Aliquots of thecDNA preparations are used in PCR amplifications with primer pairsselected from the following sets of primers: VH/JH, VK/JK or VL/JL. Theprimers used to amplify variable regions are listed in Tables 1 and 2.

[0292] (a) Heavy chain variable regions. Due to the way the plasmidexpression vectors were designed, VH primers, i.e., the forward primerin the pairs used to amplify heavy chain V regions, have the followinggeneric configuration, with the BssHII restriction site in bold:

[0293] VH primers: GCGCGCACTCC-start of VH FR1 primer.

[0294] The primers are designed to include codons encoding the last 4amino acids in the leader, with the BssHII site coding for amino acids−4 and −3 , followed by the VH family-specific FR1 sequence. Tables 1and 2 lists the sequences of the different family-specific VH primers.Since the last 5 amino acids of the heavy chain variable region, i.e.,amino acids 109-113, which are identical among the six human heavy chainJ regions, are embedded in plasmid pVHE, JH primers, i.e., the reverseprimers used to amplify the heavy chain variable regions, exhibit thefollowing configuration to include a BstEII site, which codes for aminoacids 109 and 110 (shown in bold):

[0295] JH primers:

[0296] nucleotide sequence for amino acids 103-108 of VH (ending with aG)-GTCACC

[0297] Using these sets of primers, the VH PCR products start with thecodons coding for amino acids −4 to 110 with BssHII being amino acids −4and −3, and end at the BstEII site at the codons for amino acids 109 and110 . Upon digestion with the appropriate restriction enzymes, these PCRproducts are cloned into pVHE digested with BssHII and BstEII.

[0298] In order to achieve amplification of most of the possiblerearranged heavy chain variable regions, families of VH and JH primers,as shown in Tables 1 and 2, are used. The VH1, 3, and 4 families accountfor 44 out of the 51 V regions present in the human genome. Theembedding of codons coding for amino acids 109-113 in the expressionvector precludes the use of a single common JH primer. However, the 5 JHprimers shown in Tables 1 and 2 can be pooled for each VH primer used toreduce the number of PCR reactions required.

[0299] (b) Kappa light chain variable regions. The VK primers, i.e., theforward primer in the pairs used to amplify kappa light chain variableregions, have the following generic configuration, with the ApaLIrestriction site in bold:

[0300] VK primer: GTGCACTCC-start of VK FR1 primer

[0301] The VK primers contain codons coding for the last 3 amino acidsof the kappa light chain leader with the ApaLI site coding for aminoacids −3 and −2, followed by the VK family-specific FR1 sequences. Sincethe codons encoding the last 4 amino acids of the kappa chain variableregion (amino acids 104-107) are embedded in the expression vector pVKE,the JK primers, i.e., the reverse primer in the pairs used to amplifykappa light chain variable regions, exhibit the following configuration:

[0302] JK primer:

[0303] nucleotide sequence coding for amino acids 98-103 of VK-CTCGAG

[0304] The XhoI site (shown in bold) comprises the codons coding foramino acids 104-105 of the kappa light chain variable region. The PCRproducts encoding kappa light chain variable regions start at the codonfor amino acid −3 and end at the codon for amino acid 105, with theApaLI site comprising the codons for amino acids −3 and −2 and the XhoIsite comprising the codons for amino acids 104 and 105. VK1/4 and VK3/6primers each have two degenerate nucleotide positions. Employing theseJK primers (see Tables 1 and 2), JK1, 3 and 4 will have a Val to Leumutation at amino acid 104, and JK3 will have an Asp to Glu mutation atamino acid 105.

[0305] (c) Lambda light chain variable regions. The VL primers, i.e.,the forward primer in the pairs used to amplify lambda light chainvariable regions, have the following generic configuration, with theApaLI restriction site in bold:

[0306] VL primer: GTGCACTCC-start of VL

[0307] The ApaLI site comprises the codons for amino acids −3 and −2,followed by the VL family-specific FR1 sequences. Since the codonsencoding the last 5 amino acids of VL (amino acids 103-107) are embeddedin the expression vector pVLE, the JL primers exhibit the followingconfiguration to include a HindIII site (shown in bold) comprising thecodons encoding amino acids 103-104:

[0308] JL primer: -nucleotide sequence for amino acids 97-102 ofVL-AAGCTT

[0309] The PCR products encoding lambda light chain variable regionsstart at the codon for amino acid −3 and end at the codon for amino acid104 with the ApaLI site comprising the codons for amino acids −3 and −2,and HindIII site comprising the codons for amino acids 103 and 104.TABLE 1 Oligonucleotide primers for PCR amplification of humanimmunoglobulin variable regions. Recognition sites for restrictionenzymes used in cloning are indicated in bold type. Primer sequences arefrom 5′ to 3′. VH1 (SEQ ID NO:34) TTT TGC GCG CAC TCC CAG GTG CAG CTGGTG CAG TCT GG VH2 (SEQ ID NO:144) AATA TGC GCG CAC TCC CAG GTC ACC TTGAAG GAG TCT GG VH3 (SEQ ID NO:35) TTT TGC GCG CAG TCC GAG GTG CAG CTGGTG GAG TCT GG VH4 (SEQ ID NO:36) TTT TGC GCG CAC TCC CAG GTG CAG CTGCAG GAG TCG GG VH5 (SEQ ID NO:145) AATA TGC GCG CAC TCC GAG GTG CAG CTGGTG CAG TCT G JH1 (SEQ ID NO:37) GAC GGT GAC CAG GGT GCC CTG GCC CCA JH2(SEQ ID NO:38) GAC GGT GAC CAG GGT GCC ACG GCC CCA JH3 (SEQ ID NO:39)GAC GGT GAC CAT TGT CCC TTG GCC CCA JH4/5 (SEQ ID NO:40) GAC GGT GAC CAGGGT TCC CTG GCC CCA JH6 (SEQ ID NO:41) GAC GGT GAC CGT GGT CCC TTG GCCCCA VK1 (SEQ ID NO:42) TTT GTG CAC TCC GAC ATC CAG ATG ACC CAG TCT CCVK2 (SEQ ID NO:43) TTT GTG CAC TCC GAT GTT GTG ATG ACT CAG TCT CC VK3(SEQ ID NO:44) TTT GTG CAC TCC GAA ATT GTG TTG ACC CAG TCT CC VK4 (SEQID NO:45) TTT GTG CAC TCC GAC ATC GTG ATG ACC CAG TCT CC VK5 (SEQ IDNO:46) TTT GTG CAC TCC GAA ACG ACA CTC ACG CAG TCT CC VK6 (SEQ ID NO:47)TTT GTG CAC TCC GAA ATT GTG CTG ACT CAG TCT CC JK1 (SEQ ID NO:48) GATCTC GAG CTT GGT CCC TTG GCC GAA JK2 (SEQ ID NO:49) GAT CTC GAG CTT GGTCCC CTG GCC AAA JK3 (SEQ ID NO:50) GAT CTC GAG TTT GGT CCC AGG GCC GAAJK4 (SEQ ID NO:51) GAT CTC GAG CTT GGT CCC TCC GCC GAA JK5 (SEQ IDNO:52) AAT CTC GAG TCG TGT CCC TTG GCC GAA VL1 (SEQ ID NO:53) TTT GTGCAC TCC CAG TCT GTG TTG ACG CAG CCG CC VL2 (SEQ ID NO:54) TTT GTG CACTCC CAG TCT GCC CTG ACT CAG CCT GC VL3A (SEQ ID NO:55) TTT GTG CAC TCCTCC TAT GTG CTG ACT CAG CCA CC VL3B (SEQ ID NO:56) TTT GTG CAC TCC TCTTCT GAG CTG ACT CAG GAC CC VL4 (SEQ ID NO:57) TTT GTG CAC TCC CAC GTTATA CTG ACT CAA CCG CC VL5 (SEQ ID NO:58) TTT GTG CAC TCC CAG GCT GTGCTC ACT CAG CCG TC VL6 (SEQ ID NO:59) TTT GTG CAC TCC AAT TTT ATG CTGACT CAG CCC CA VL7 (SEQ ID NO:60) TTT GTG CAC TCC CAG GCT GTG GTG ACTCAG GAG CC JL1 (SEQ ID NO:61) GGT AAG CTT GGT CCC AGT TCC GAA GAC JL2/3(SEQ ID NO:62) GGT AAG CTT GGT CCC TCC GCC GAA T

[0310] TABLE 2 Oligonucleotide primers for PCR amplification of humanimmunoglobulin variable regions. Recognition sites for restrictionenzymes used in cloning are indicated in bold type. Primer sequences arefrom 5′ to 3′. VH1a (SEQ ID NO:63) AATA TGC GCG CAC TCC CAG GTG CAG CTGGTG CAG TCT GG VH2a (SEQ ID NO:64) AATA TGC GCG CAC TCC CAG GTC ACC TTGAAG GAG TCT GG VH3a (SEQ ID NO:65) AATA TGC GCG CAC TCC GAG GTG CAG CTGGTG GAG TCT GG VH4a (SEQ ID NO:66) AATA TGC GCG CAC TCC CAG GTG CAG CTGCAG GAG TCG GG VH5a (SEQ ID NO:67) AATA TGC GCG CAC TCC GAG GTG CAG CTGGTG CAG TCT G JH1a (SEQ ID NO:68) GA GAC GGT GAC CAG GGT GCC CTG GCC CCAJH2a (SEQ ID NO:69) GA GAC GGT GAC CAG GGT GCC ACG GCC CCA JH3a (SEQ IDNO:70) GA GAC GGT GAC CAT TGT CCC TTG GCC CCA JH4/5a (SEQ ID NO:71) GAGAC GGT GAC CAG GGT TCC CTG GCC CCA JH6a (SEQ ID NO:72) GA GAC GGT GACCGT GGT CCC TTG GCC CCA VK1a (SEQ ID NO:73) CAGGA GTG CAC TCC GAC ATCCAG ATG ACC CAG TCT CC VK2a (SEQ ID NO:74) CAGGA GTG CAC TCC GAT GTT GTGATG ACT CAG TCT CC VK3a (SEQ ID NO:75) CAGGA GTG CAC TCC GAA ATT GTG TTGACG CAG TCT CC VK4a (SEQ ID NO:76) CAGGA GTG CAC TCC GAC ATC GTG ATG ACCCAG TCT CC VK5a (SEQ ID NO:77) CAGGA GTG CAC TCC GAA ACG ACA CTC ACG CAGTCT CC VK6a (SEQ ID NO:78) CAGGA GTG CAC TCC GAA ATT GTG CTG ACT CAG TCTCC JK1a (SEQ ID NO:79) TT GAT CTC GAG CTT GGT CCC TTG GCC GAA JK2a (SEQID NO:80) TT GAT CTC GAG CTT GGT CCC CTG GCC AAA JK3a (SEQ ID NO:81) TTGAT CTC GAG TTT GGT CCC AGG GCC GAA JK4a (SEQ ID NO:82) TT GAT CTC GAGCTT GGT CCC TCC GCC GAA JK5a (SEQ ID NO:83) TT AAT CTC GAG TCG TGT CCCTTG GCC GAA VL1a (SEQ ID NO:84) CAGAT GTG CAC TCC CAG TCT GTG TTG ACGCAG CCG CC VL2a (SEQ ID NO:85) CAGAT GTG CAC TCC CAG TCT GCC CTG ACT CAGCCT GC VL3Aa (SEQ ID NO:86) CAGAT GTG CAC TCC TCC TAT GTG CTG ACT CAGCCA CC VL3Ba (SEQ ID NO:87) CAGAT GTG CAC TCC TCT TCT GAG CTG ACT CAGGAC CC VL4a (SEQ ID NO:88) CAGAT GTG CAC TCC CAC GTT ATA CTG ACT CAA CCGCC VL5a (SEQ ID NO:89) CAGAT GTG CAC TCC CAG GCT GTG CTC ACT CAG CCG TCVL6a (SEQ ID NO:90) CAGAT GTG CAC TCC AAT TTT ATG CTG ACT CAG CCC CAVL7a (SEQ ID NO:91) CAGAT GTG CAC TCC CAG GCT GTG GTG ACT CAG GAG CCJL1a (SEQ ID NO:92) AC GGT AAG CTT GGT CCC AGT TCC GAA GAC JL2/3a (SEQID NO:93) AC GGT AAG CTT GGT CCC TCC GCC GAA TAC

Example 2 Strategies for Selection of Human Immunoglobulins Which Bind aSpecific Antigen

[0311] Vaccinia virus expression vectors comprising polynucleotidesencoding recombinant heavy chain immunoglobulin subunit polypeptideswhich, in combination with some unidentified light chain, conferspecificity for a defined antigen, are selected as follows, and as shownin FIG. 1. Selection of specific immunoglobulin heavy and light chainsis accomplished in two phases. First, a library of diverse heavy chainsfrom antibody producing cells of either naive or immunized donors isconstructed in a pox virus based vector by trimolecular recombination(see Example 5) using as a transfer plasmid pVHE, constructed asdescribed in Example 1, and a similarly diverse library ofimmunoglobulin light chains is constructed in a plasmid vector such aspVKE and pVLE, constructed as described in Example 1, in whichexpression of the recombinant gene is regulated by the p7.5 vacciniapromoter. The immunoglobulin heavy chain constant region in the poxvirus constructs is designed to retain the transmembrane region thatresults in expression of immunoglobulin receptor on the surfacemembrane. Host cells, e.g., early B cell lymphoma cells, are infectedwith the pox virus heavy chain library at a multiplicity of infection of1 (MOI=1). Two hours later the infected cells are transfected with thelight chain plasmid library under conditions which allow, on average, 10or more separate light chain plasmids to be taken up and expressed ineach cell. Because expression of the recombinant gene in this plasmid isregulated by a vaccinia virus promoter, high levels of the recombinantgene product are expressed in the cytoplasm of vaccinia virus infectedcells without a requirement for nuclear integration. Under theseconditions a single cell can express multiple antibodies with differentlight chains associated with the same heavy chains in characteristicH₂L₂ structures in each infected cell.

[0312] 2.1 Direct antigen-induced apoptosis. An early B celllymphomahost cell is infected with recombinant vaccinia viruses encodingrecombinant heavy chain immunoglobulin subunit polypeptides andtransfected with plasmids encoding recombinant light chainimmunoglobulin subunit polypeptides as described. The host cells respondto crosslinking of antigen-specific immunoglobulin receptors byinduction of spontaneous growth inhibition and apoptotic cell death. Asoutlined in FIG. 1, synthesis and assembly of antibody molecules isallowed to proceed for 12 hours or more at which time specific antigenis presented on a synthetic particle or polymer, or on the surface of anantigen expressing cell, in order to crosslink any specificimmunoglobulin receptors and induce apoptosis of selected antibodyexpressing indicator cells. The genomes of recombinant vaccinia virusesextracted from cells in which apoptosis has been induced are enrichedfor polynucleotides encoding immunoglobulin heavy chain genes thatconfer the desired specificity.

[0313] 2.2 Indirect antigen-induced cell death. As shown in FIG. 2A(bottom) and FIG. 2B (top), an early B cell lymphoma host cell istransfected with a construct in which the promoter of an apoptosisinduced gene, here, a BAX promoter, drives expression of a foreigncytotoxic T cell epitope. The host cells express the CTL epitope inresponse to crosslinking of antigen-specific immunoglobulin receptors,and these cross-linked cells will undergo a lytic event upon theaddition of specific CTL. The stably transfected host cells are theninfected with recombinant vaccinia viruses encoding recombinant heavychain immunoglobulin subunit polypeptides and transfected with plasmidsencoding recombinant light chain immunoglobulin subunit polypeptides asdescribed. As outlined in FIG. 1, synthesis and assembly of antibodymolecules is allowed to proceed for 12 hours or more at which timespecific antigen is presented on a synthetic particle or polymer, or onthe surface of an antigen expressing cell, in order to cross-link anyspecific immunoglobulin receptors. Upon addition of epitope-specificCTL, those cells in which surface immunoglobulin molecules are crosslinked undergo a lytic event, thereby indirectly inducing cell death.

[0314] 2.3 Direct antigen-induced cell death. As shown in FIG. 2A (top)and FIG. 2B (bottom), an early B cell lymphoma host cell is transfectedwith a construct in which the promoter of an apoptosis induced gene,here, a BAX promoter, drives expression of the cytotoxic A subunit ofdiphtheria toxin. The host cells express the toxin subunit in responseto cross linking of antigen-specific immunoglobulin receptors, and thesecross-linked cells will succumb to cell death. The stably transfectedhost cells are then infected with recombinant vaccinia viruses encodingrecombinant heavy chain immunoglobulin subunit polypeptides andtransfected with plasmids encoding recombinant light chainimmunoglobulin subunit polypeptides as described. As outlined in FIG. 1,synthesis and assembly of antibody molecules is allowed to proceed for12 hours or more at which time specific antigen is presented on asynthetic particle or polymer, or on the surface of an antigenexpressing cell, in order to cross-link any specific immunoglobulinreceptors. Those cells in which surface immunoglobulin molecules arecross linked rapidly and directly succumb to cell death.

[0315] 2.4 Discussion. The reason expression of these recombinant genesis upregulated by crosslinking surface Ig receptors is that expressionof each of the two constructs is regulated by the promoter for a genewhose expression is naturally upregulated in early B cell lymphoma cellsfollowing Ig crosslinking. This is illustrated by use of the BAXpromoter. BAX being an example of a proapoptotic gene that is normallyupregulated in early B cell lymphoma cells under these conditions.Regulatory regions (the “promoter”) for other genes may serve equallywell or better. Such genes are identified, for example, by comparing thegene expression profile of early B cell lymphoma cells on microarraysbefore and after crosslinking of membrane Ig.

[0316] Cells are transfected with a construct leading to expression ofthe diphtheria A chain (dipA), undergo more rapid apoptosis than isinduced by Ig crosslinking alone. An even more rapid cell death isinduced by addition of cytotoxic T cells specific for some targetpeptide that associates with a native MHC molecule expressed in thatcell and that is encoded by a minigene whose expression is regulated bya BAX or BAX-like promoter. In addition, host cells other than early Bcell lymphoma cells are likewise engineered to express genes whicheither directly or indirectly induce cell death upon antigen crosslinking of surface immunoglobulin molecules, independent of theprogrammed apoptosis which occurs in early B cell lymphoma cell linesupon antigen cross linking.

[0317] A variety of substrates are employed to present antigen andcross-link specific membrane immunoglobulin receptors in the aboveselection process. These include, but are not limited to, magneticbeads, protein coated tissue culture plates, and cells transfected witha gene encoding the target antigen. Examples of cells that may betransfected for efficient expression of the target antigen include, butare not limited to, L cells and NIH 3T3 cells. However, if a transfectedcell is employed to express and present a recombinant antigen, then isnecessary to first deplete the immunoglobulin-expressing host cellpopulation of any host cells that express antibodies reactive withmembrane antigens of the non-transfected cell. Such depletion could beaccomplished in one or more rounds of absorption to non-transfectedcells bound to a solid substrate. It would then be possible to employthe antigen expressing transfectant for positive selection of cellsexpressing specific recombinant antibodies. In a preferred embodiment,alternating cycles of negative and positive selection are repeated asoften as necessary to achieve a desired enrichment.

[0318] In one example of a positive selection step, antibody expressingB lymphoma cells are allowed to adhere to a solid substrate to which Bcell specific anti-CD19 and/or anti-CD20 antibody has been bound.Adherent indicator cells that undergo a lytic event are induced torelease their cytoplasmic contents including any viral immunoglobulinheavy chain recombinants into the culture fluid. Recombinant virusesharvested from cells and cell fragments recovered in the culture fluidare enriched for those recombinant viruses that encode an immunoglobulinheavy chain which confers specificity for the selecting antigen whenassociated with some as yet unidentified light chains. Additional cyclesof antigen driven selection in cells freshly infected with this enrichedpopulation of recombinant viruses and subsequently transfected with thesame initial population of unselected plasmids encoding diverse lightchains leads to further enrichment of the desired heavy chains.Following multiple reiterations of this selection process, a smallnumber of heavy chains are isolated which possess optimal specificityfor a defined antigen when associated with some unidentified lightchains.

[0319] In order to select light chains that confer the desiredspecificity in association with the previously selected heavy chains,the entire selection process as described above is repeated by infectinghost cells at MOI=1 with a library of diverse light chain recombinantsin the vaccinia based vector followed by transfection with a plasmidrecombinant for one of the previously selected heavy chains. The optimallight chain partners for that heavy chain are isolated followingmultiple cycles of antigen driven selection as described above.

[0320] In another preferred embodiment, a similar strategy isimplemented by exploiting the binding properties conferred on a cellthat expresses specific antibody on its surface membrane. Instead ofemploying early B cell lymphomas that undergo apoptosis in response toreceptor crosslinking as indicator cells, this strategy, depicted inFIG. 5, allows host cells expressing a desired immunoglobulinspecificity to be selected by binding to synthetic particles or polymersto which antigen is coupled or to the surface of a specific antigenexpressing transfected cell. In this case the indicator cells are chosenfor the ability to express high levels of membrane immunoglobulinreceptors rather than for an apoptotic response to crosslinking ofmembrane immunoglobulin receptors. Preferred cell lines includeimmunoglobulin negative plasmacytomas. Other issues related to thespecificity, background and efficiency of the selection process aretreated as described above.

Example 3 Selection of an Antibody with Defined Specificity from aLibrary of 10⁹ Combinations of Immunoglobulin Heavy and Light Chains

[0321] The affinity of specific antibodies that can be selected from alibrary is a function of the size of that library. In general, thelarger the number of heavy and light chain combinations represented inthe library, the greater the likelihood that a high affinity antibody ispresent and can be selected. Previous work employing phage displaymethods has suggested that for many antigens a library that includes 10⁹immunoglobulin heavy and light chain combinations is of a sufficientsize to select a relatively high affinity specific antibody. Inprinciple, it is possible to construct a library with 10⁹ recombinantseach of which expresses a unique heavy chain and a unique light chain ora single chain construct with a combining site comprising variableregions of heavy and light chains. The most preferred method, however,is to generate this number of antibody combinations by constructing twolibraries of 10⁵ immunoglobulin heavy chains and 10⁴ immunoglobulinlight chains that can be co-expressed in all 10⁹ possible combinations.In this example greater diversity is represented in the heavy chain poolbecause heavy chains have often been found to make a greatercontribution than the associated light chain to a specific antigencombining site.

[0322] 3.1 Heavy Chain Genes. A library of vaccinia recombinants at atiter of approximately 10⁶ is constructed from a minimum of 10⁵immunoglobulin heavy chain cDNA transfer plasmid recombinantssynthesized by the methods previously described (Example 1) from RNAderived from a pool of 100 bone marrow donors. As described below, thislibrary must be further expanded to a titer of at least 10⁹ heavy chainrecombinants. A preferred method to expand the library is to infectmicrocultures of approximately 5×10⁴ BSC1 cells with individual pools of10³ vaccinia heavy chain recombinants. Typically a greater than 1,000fold expansion in the viral titer is obtained after 48 hrs infection.Expanding viral titers in multiple individual pools mitigates the riskthat a subset of recombinants will be lost due to relatively rapidgrowth of a competing subset.

[0323] 3.2 Light Chain Genes. A library of vaccinia recombinants at atiter of approximately 10⁵ is constructed from a minimum of 10⁴immunoglobulin light chain cDNA transfer plasmid recombinantssynthesized from RNA derived from a pool of bone marrow donors asdescribed in Example 1. For use in multiple cycles of heavy chainselection as described below, this library must be further expanded to atiter of 10¹⁰ to 10¹¹ light chain recombinants. A preferred method toexpand the library is to infect 100 microcultures of approximately 5×10⁴BSC1 cells with individual pools of 10³ vaccinia light chainrecombinants. Viral recombinants recovered from each of the 100 infectedcultures are further expanded as a separate pool to a titer of between10⁸ and 10⁹ viral recombinants. It is convenient to label these lightchain pools L1 to L100.

[0324] 3.3 Selection of Immunoglobulin Heavy Chain Recombinants. 100cultures of 10⁷ cells of a non-producing myeloma, preferably Sp2/0, orearly B cell lymphoma, preferably CH33, are infected with viablevaccinia heavy chain recombinants at MOI=1 and simultaneously withpsoralen (4′-aminomethyl-Trioxsalen) inactivated vaccinia light chainrecombinants at MOI=1 to 10 (see below). For psoralen inactivation,cell-free virus at 10⁸ to 10⁹ pfu/ml is treated with 10 μg/ml psoralenfor 10 minutes at 25° C. and then exposed to long-wave (365-nm) UV lightfor 2 minutes (Tsung, K., J. H. Yim, W. Marti, R. M. L. Buller, and J.A. Norton. J. Virol. 70:165-171 (1996)) The psoralen treated virus isunable to replicate but allows expression of early viral genes includingrecombinant genes under the control of early but not late viralpromoters. Under these conditions, light chains synthesized frompsoralen treated recombinants will be assembled into immunoglobulinmolecules in association with the single heavy chain that is, onaverage, expressed in each infected cell.

[0325] The choice of infection with psoralen inactivated light chainrecombinants at MOI=1 or at MOI=10 will influence the relativeconcentration in a single positive cell of a particular H+L chaincombination which will be high at MOI=1 and low (because of dilution bymultiple light chains) at MOI=10. A low concentration andcorrespondingly reduced density of specific immunoglobulin at the cellsurface is expected to select for antibodies with higher affinity forthe ligand of interest. On the other hand, a high concentration ofspecific receptor is expected to facilitate binding or signaling throughthe immunoglobulin receptor.

[0326] Following a first cycle of antigen-specific selection by bindingor signaling as described in Example 2, an enriched population ofrecombinant virus is recovered from each culture with a titer which,during this initial selection and depending on background levels ofnon-specific binding or spontaneous release of virus, may be between 1%and 10% of the titer of input virus. It is convenient to label as H1a toH100a the heavy chain recombinant pools recovered from cultures in thefirst cycle of selection that received psoralen treated virus from theoriginal light chain recombinant pools L1 to L100 respectively.

[0327] To carry out a second cycle of selection under the sameconditions as the first cycle, it is again necessary to expand the titerof recovered heavy chain recombinants by 10 to 100 fold. For the secondcycle of selection non-producing myeloma or early B cell lymphoma areagain infected with viable viral heavy chain recombinants and psoralentreated light chain recombinants such that, for example, the sameculture of 10⁷ cells is infected with heavy chain recombinants recoveredin pool H37a and psoralen treated light chain recombinants from theoriginal L37 pool employed to select H37a. Heavy chain recombinantsrecovered from the H37a pool in the second cycle of selection areconveniently labeled H37b and so on.

[0328] Following the second cycle of selection, specific viralrecombinants are likely, in general, to be enriched by a factor of 10 ormore relative to the initial virus population. In this case, it is notnecessary for the third cycle of selection to be carried out under thesame conditions as the first or second cycle since specific clones arelikely to be well-represented even at a 10 fold lower titer. For thethird cycle of selection, therefore, 100 cultures of only 10⁶non-producing myeloma or early B cell lymphoma are again infected withviable viral heavy chain recombinants and psoralen treated light chainrecombinants from cognate pools. Another reduction by a factor of 10 inthe number of infected cells is effected after the 5th cycle ofselection.

[0329] 3.4 Identification of Antigen-specific Heavy Chain Recombinants.

[0330] (a) Following any given cycle of selection it is possible todetermine whether antigen-specific heavy chains have been enriched to alevel of 10% or more in a particular pool, for example H37f, by picking10 individual viral pfu from that heavy chain pool to test forantigen-specificity in association with light chains of the original L37pool. Since the light chain population comprises 10⁴ diverse cDNAdistributed among 100 individual pools, the average pool hasapproximately 10² different light chains. Even if a selected heavy chainconfers a desired antigenic specificity only in association with asingle type of light chain in the available light chain pool, 1% ofcells infected with the selected heavy chain recombinant and the randomlight chain pool at MOI=1 will express the desired specificity. Thisfrequency can be increased to 10% on average if cells are infected withlight chains at MOI=10. A preferred method to confirm specificity is toinfect with immunoglobulin heavy chain and a pool of light chains a lineof CH33 early B cell lymphoma transfected with an easily detectedreporter construct, for example luciferase, driven by the promoter forBAX or another CH33 gene that is activated as a result of membranereceptor crosslinking. Infection of this transfectant with the plaquepurified heavy chain recombinant and the relevant light chain pool willresult in an easily detected signal if the selected heavy chain confersthe desired antigenic specificity in association with any of the 100 ormore light chains represented in that pool. Note that this same methodis applicable to analysis of heavy chains whether they are selected byspecific-binding or by specific-signaling through immunoglobulinreceptors of infected cells.

[0331] (b) An alternative method to identify the most promisingantigen-specific heavy chains is to screen for those that are mosthighly represented in the selected population. Inserts can be isolatedby PCR amplification with vector specific primers flanking the insertionsite and these inserts can be sequenced to determine the frequency ofany observed sequence. In this case, however, it remains necessary toidentify a relevant light chain as described below.

[0332] 3.5 Selection of Immunoglobulin Light Chain Recombinants. Once anantigen-specific heavy chain has been isolated, a light chain thatconfers antigen-specificity in association with that heavy chain can beisolated from the pool that was employed to select that heavy chain asdescribed in 3.4(a). Alternatively, it may be possible to select yetanother light chain from a larger library that, in association with thesame heavy chain, could further enhance affinity. For this purpose alibrary of vaccinia recombinants at a titer of approximately 10⁶ isconstructed from a minimum of 10⁵ immunoglobulin light chain cDNAtransfer plasmid recombinants synthesized by the methods previouslydescribed (Example 1). The procedure described in 3.3 is reversed suchthat non-producing myeloma or early B cell lymphoma are now infectedwith viable viral light chain recombinants at MOI=1 and a singleselected psoralen treated specific heavy chain recombinant. To promoteselection of higher affinity immunoglobulin, it may be preferable todilute the concentration of each specific H+L chain pair by infectionwith light chains at MOI=10.

[0333] 3.6 Selection of Immunoglobulin Heavy Chain Recombinants in thePresence of a Single Immunoglobulin Light Chain. The selection of animmunoglobulin heavy chain that can contribute to a particular antibodyspecificity is simplified if a candidate light chain has already beenidentified. This may be the case if, for example a murine monoclonalantibody has been previously selected. The murine light chain variableregion can be grafted to a human light chain constant region to optimizepairing with human heavy chains, a process previously described byothers employing phage display methods as “Guided Selection” (Jespers,L. S., A. Roberts, S. M. Mahler, G. Winter, H. R. and Hoogenboom.Bio/Technology 12:899-903, 1994; Figini, M., L. Obici, D. Mezzanzanica,A. Griffiths, M. I. Colnaghi, G. Winters, and S. Canevari. Cancer Res.58:991-996, 1998). This molecular matching can, in principle, be takeneven further if human variable gene framework regions are also graftedinto the murine light chain variable region sequence (Rader, C., D. A.Cheresh, and C. F. Barbas III. Proc. Natl. Acad. Sci. USA 95:8910-8915).Any human heavy chains selected to pair with this modifiedantigen-specific light chain can themselves become the basis forselection of an optimal human light chain from a more diverse pool asdescribed in 3.5.

Example 4 Selection of Specific Human Antibodies from a cDNA LibraryConstructed in Adenovirus, Herpesvirus, or Retrovirus vectors

[0334] 4.1 Herpesvirus. A method has been described for the generationof helper virus free stocks of recombinant, infectious Herpes SimplexVirus Amplicons (T. A. Stavropoulos, C. A. Strathdee. 1998 J.Virology72:7137-7143). It is possible that a cDNA Library of humanImmunoglobulin Heavy and/or Light chain genes or fragments thereof,including single chain fragments, constructed in the plasmid Ampliconvector could be packaged into a library of infectious amplicon particlesusing this method. An Amplicon library constructed using immunoglobulinheavy chain genes, and another Amplicon library constructed usingimmunoglobulin light chain genes could be used to coinfect anon-producing myeloma cell line. The myeloma cells expressing animmunoglobulin gene combination with the desired specificity can beenriched by selection for binding to the antigen of interest. The HerpesAmplicons are capable of stable transgene expression in infected cells.Cells selected for binding in a first cycle will retain theirimmunoglobulin gene combination, and will stably express antibody withthis specificity. This allows for the reiteration of selection cyclesuntil immunoglobulin genes with the desired specificity can be isolated.Selection strategies that result in cell death could also be attempted.The amplicon vector recovered from these dead selected cells cannot beused to infect fresh target cells, because in the absence of helpervirus the amplicons are replication defective and will not be packagedinto infectious form. The amplicon vectors contain a plasmid origin ofreplication and an antibiotic resistance gene. This makes it possible torecover the selected amplicon vector by transforming DNA purified fromthe selected cells into bacteria. Selection with the appropriateantibiotic would allow for the isolation of bacterial cells that hadbeen transformed by the amplicon vector. The use of different antibioticresistance genes on the heavy and light chain Amplicon vectors, forexample ampicillin and kanamycin, would allow for the separate selectionof heavy and light chain genes from the same population of selectedcells. Amplicon plasmid DNA can be extracted from the bacteria andpackaged into infectious viral particles by cotransfection of theamplicon DNA and packaging defective HSV genomic DNA into packagingcells. Infectious amplicon particles can then be harvested and used toinfect a fresh population of target cells for another round of selection

[0335] 4.2 Adenovirus. Methods have been described for the production ofrecombinant Adenovirus (S. Miyake, M. Makimura, Y. Kanegae, S. Harada,Y. Sato, K. Takamori, C. Tokuda, I. Saito. 1996 Proc. Natl. Acad. Sci.USA 93: 1320-1324; T. C. He, S. Zhou, L. T. Da Costa, J. Yu, K. W.Kinzler, B. Volgelstein. 1998 Proc. Natl. Acad. Sci. USA 95: 2509-2514)It is possible that a cDNA library could be constructed in an Adenovirusvector using either of these methods. Insertion of cDNA into the E3 orE4 region of Adenovirus results in a replication competent recombinantvirus. This library could be used for similar applications as thevaccinia cDNA libraries constructed by trimolecular recombination. Forexample a heavy chain cDNA library can be inserted into the E3 or E4region of Adenovirus. This results in a replication competent heavychain library. A light chain cDNA library could be inserted into the E1gene of Adenovirus, generating a replication defective library. Thisreplication defective light chain library can be amplified by infectionof cells that provide Adenovirus E1 in trans, such as 293 cells. Thesetwo libraries can be used in similar selection strategies as thosedescribed using replication competent vaccinia heavy chain library andPsoralen inactivated vaccinia light chain library.

[0336] 4.3 Advantages of vaccinia virus. Vaccinia virus possessesseveral advantages over Herpes or Adenovirus for construction of cDNALibraries. First, vaccinia virus replicates in the cytoplasm of the hostcell, while HSV and Adenovirus replicate in the nucleus. A higherfrequency of cDNA recombinant transfer plasmid may be available forrecombination in the cytoplasm with vaccinia than is able to translocateinto the nucleus for packaging/recombination in HSV or Adenovirus.Second, vaccinia virus, but not Adenovirus or Herpes virus, is able toreplicate plasmids in a sequence independent manner (M. Merchlinsky, B.Moss. 1988 Cancer Cells 6: 87-93). Vaccinia replication of cDNArecombinant transfer plasmids may result in a higher frequency ofrecombinant virus being produced. Although we have described thepotential construction of cDNA Libraries in Herpes or Adenovirusvectors, it should be emphasized that there has been no reported use ofthese methods to construct a cDNA Library in either of these viralvectors.

[0337] 4.4 Retrovirus. Construction of cDNA Libraries in replicationdefective retroviral vectors have been described (T. Kitamura, M.Onishi, S. Kinoshita, A. Shibuya, A. Miyajima, and G. P. Nolan. 1995PNAS 92:9146-9150; I. Whitehead, H. Kirk, and R. Kay. 1995 Molecular andCellular Biology 15:704-710.). Retroviral vectors integrate uponinfection of target cells, and have gained widespread use for theirability to efficiently transduce target cells, and for their ability toinduce stable transgene expression. A Retroviral cDNA libraryconstructed using immunoglobulin heavy chain genes, and anotherRetroviral library constructed using immunoglobulin light chain genescould be used to coinfect a non-producing myeloma cell line. The myelomacells expressing an immunoglobulin gene combination with the desiredspecificity can be enriched for by selection for binding to the antigenof interest. Cells selected for binding in a first cycle will retaintheir immunoglobulin gene combination, and will stably expressimmunoglobulins with this specificity. This allows for the reiterationof selection cycles until immunoglobulin genes with the desiredspecificity can be isolated.

Example 5 Trimolecular Recombination

[0338] 5.1 Production of an Expression Library. This example describes atri-molecular recombination method employing modified vaccinia virusvectors and related transfer plasmids that generates close to 100%recombinant vaccinia virus and, for the first time, allows efficientconstruction of a representative DNA library in vaccinia virus. Thetrimolecular recombination method is illustrated in FIG. 6.

[0339] 5.2 Construction of the Vectors. The previously describedvaccinia virus transfer plasmid pJ/K, a pUC 13 derived plasmid with avaccinia virus thymidine kinase gene containing an in-frame Not I site(Merchlinsky, M. et al., Virology 190:522-526), was further modified toincorporate a strong vaccinia virus promoter followed by Not I and Apa Irestriction sites. Two different vectors, p7.5/tk and pEL/tk, included,respectively, either the 7.5K vaccinia virus promoter or a strongsynthetic early/late (E/L) promoter (FIG. 7). The Apa I site waspreceded by a strong translational initiation sequence including the ATGcodon. This modification was introduced within the vaccinia virusthymidine kinase (tk) gene so that it was flanked by regulatory andcoding sequences of the viral tk gene. The modifications within the tkgene of these two new plasmid vectors were transferred by homologousrecombination in the flanking tk sequences into the genome of theVaccinia Virus WR strain derived vNotI⁻vector to generate new viralvectors v7.5/tk and vEL/tk. Importantly, following Not I and ApaIrestriction endonuclease digestion of these viral vectors, two largeviral DNA fragments were isolated each including a separatenon-homologous segment of the vaccinia tk gene and together comprisingall the genes required for assembly of infectious viral particles.Further details regarding the construction and characterization of thesevectors and their alternative use for direct ligation of DNA fragmentsin vaccinia virus are described in Example 1.

[0340] 5.3 Generation of an Increased Frequency of Vaccinia VirusRecombinants. Standard methods for generation of recombinants invaccinia virus exploit homologous recombination between a recombinantvaccinia transfer plasmid and the viral genome. Table 3 shows theresults of a model experiment in which the frequency of homologousrecombination following transfection of a recombinant transfer plasmidinto vaccinia virus infected cells was assayed under standardconditions. To facilitate functional assays, a minigene encoding theimmunodominant 257-264 peptide epitope of ovalbumin in association withH-2K^(b) was inserted at the Not 1 site in the transfer plasmid tk gene.As a result of homologous recombination, the disrupted tk gene issubstituted for the wild type viral tk+ gene in any recombinant virus.This serves as a marker for recombination since tk− human 143B cellsinfected with tk− virus are, in contrast to cells infected with wildtype tk+ virus, resistant to the toxic effect of BrdU. Recombinant viruscan be scored by the viral pfu on 143B cells cultured in the presence of125 mM BrdU.

[0341] The frequency of recombinants derived in this fashion is of theorder of 0.1% (Table 3). TABLE 3 Generation of Recombinant VacciniaVirus by Standard Homologous Recombination Titer w/o Titer w/ % Virus*DNA BrdU BrdU Recombinant** vaccinia — 4.6 × 10⁷ 3.0 × 10³ 0.006vaccinia 30 ng pE/Lova 3.7 × 10⁷ 3.2 × 10⁴ 0.086 vaccinia 300 ng pE/Lova2.7 × 10⁷ 1.5 × 10⁴ 0.056

[0342] This recombination frequency is too low to permit efficientconstruction of a cDNA library in a vaccinia vector. The following twoprocedures were used to generate an increased frequency of vacciniavirus recombinants.

[0343] (1) One factor limiting the frequency of viral recombinantsgenerated by homologous recombination following transfection of aplasmid transfer vector into vaccinia virus infected cells is that viralinfection is highly efficient whereas plasmid DNA transfection isrelatively inefficient. As a result many infected cells do not take uprecombinant plasmids and are, therefore, capable of producing only wildtype virus. In order to reduce this dilution of recombinant efficiency,a mixture of naked viral DNA and recombinant plasmid DNA was transfectedinto Fowl Pox Virus (FPV) infected mammalian cells. As previouslydescribed by others (Scheiflinger, F., et al., 1992, Proc. Natl. Acad.Sci. USA 89:9977-9981), FPV does not replicate in mammalian cells butprovides necessary helper functions required for packaging maturevaccinia virus particles in cells transfected with non-infectious nakedvaccinia DNA. This modification of the homologous recombinationtechnique alone increased the frequency of viral recombinantsapproximately 35 fold to 3.5% (Table 4). TABLE 4 Generation ofRecombinant Vaccinia Virus by Modified Homologous Recombination Titerw/o Titer w/ % Virus DNA BrdU BrdU Recombinant* PFV None 0 0 0 Nonevaccinia WR 0 0 0 PFV vaccinia WR 8.9 × 10⁶ 2.0 × 10² 0.002 PFV vacciniaWR + 5.3 × 10⁶ 1.2 × 10⁵ 2.264 pE/Lova (1:1) PFV vaccinia WR + 8.4 × 10⁵3.0 × 10⁴ 3.571 pE/Lova (1:10)

[0344] Table 4. Confluent monolayers of BSC1 cells (5×10⁵ cells/well)were infected with moi=1.0 of fowlpox virus strain HP1. Two hours latersupernatant was removed, cells were washed 2× with Opti-Mem I media, andtransfected using lipofectamine with 600 ng vaccinia strain WR genomicDNA either alone, or with 1:1 or 1:10 (vaccinia:plasmid) molar ratios ofplasmid pE/Lova. This plasmid contains a fragment of the ovalburnincDNA, which encodes the SIINFEKL epitope, known to bind with highaffinity to the mouse class I MHC molecule K^(b). Expression of thisminigene is controlled by a strong, synthetic Early/Late vacciniapromoter. This insert is flanked by vaccinia tk DNA. Three days latercells were harvested, and virus extracted by three cycles of freeze/thawin dry ice isopropanol/37° C. water bath. Crude virus stocks weretitered by plaque assay on human TK-143B cells with and without BrdU.

[0345] (2) A further significant increase in the frequency of viralrecombinants was obtained by transfection of FPV infected cells with amixture of recombinant plasmids and the two large approximately 80kilobases and 100 kilobases fragments of vaccinia virus v7.5/tk DNAproduced by digestion with NotI and ApaI restriction endonucleases.Because the NotI and Apa I sites have been introduced into the tk gene,each of these large vaccinia DNA arms includes a fragment of the tkgene. Since there is no homology between the two tk gene fragments, theonly way the two vaccinia arms can be linked is by bridging through thehomologous tk sequences that flank the inserts in the recombinanttransfer plasmid. The results in Table 5 show that >99% of infectiousvaccinia virus produced in triply transfected cells is recombinant for aDNA insert as determined by BrdU resistance of infected tk− cells. TABLE5 Generation of 100% Recombinant Vaccinia Virus Using Tri-MolecularRecombination Titer w/o Titer w/ % Virus DNA BrdU BrdU Recombinant* PFVUncut v7.5/tk 2.5 × 10⁶ 6.0 × 10³ 0.24 PFV NotI/Apal v7.5/tk arms 2.0 ×10² 0 0 PFV NotI/Apal v7.5/tk arms + 6.8 × 10⁴ 7.4 × 10⁴ 100 pE/Lova(1:1)

[0346] Table 5. Genomic DNA from vaccinia strain V7.5/tk (1.2micrograms) was digested with ApaI and NotI restriction endonucleases.The digested DNA was divided in half. One of the pools was mixed with a1:1 (vaccinia:plasmid) molar ratio of pE/Lova. This plasmid contains afragment of the ovalbumin cDNA, which encodes the SIINFEKL epitope,known to bind with high affinity to the mouse class I MHC moleculeK^(b). Expression of this minigene is controlled by a strong, syntheticEarly/Late vaccinia promoter. This insert is flanked by vaccinia tk DNA.DNA was transfected using lipofectamine into confluent monolayers (5×10⁵cells/well) of BSC1 cells, which had been infected 2 hours previouslywith moi=1.0 FPV. One sample was transfected with 600 ng untreatedgenomic V7.5/tk DNA. Three days later cells were harvested, and thevirus was extracted by three cycles of freeze/thaw in dry iceisopropanol/37° C. water bath. Crude viral stocks were plaqued on TK-143B cells with and without BrdU selection.

[0347] 5.4 Construction of a Representative cDNA Library in VacciniaVirus. A cDNA library is constructed in the vaccinia vector todemonstrate representative expression of known cellular mRNA sequences.Additional modifications have been introduced into the p7.5/tk transferplasmid and v7.5/tk viral vector to enhance the efficiency ofrecombinant expression in infected cells. These include introduction oftranslation initiation sites in three different reading frames and ofboth translational and transcriptional stop signals as well asadditional restriction sites for DNA insertion.

[0348] First, the HindIII J fragment (vaccinia tk gene) of p7.5/tk wassubcloned from this plasmid into the HindIII site of pBS phagemid(Stratagene) creating pBS.Vtk.

[0349] Second, a portion of the original multiple cloning site ofpBS.Vtk was removed by digesting the plasmid with SmaI and PstI,treating with Mung Bean Nuclease, and ligating back to itself,generating pBS.Vtk.MCS-. This treatment removed the unique SmaI, BamHI,SalI, and PstI sites from pBS.Vtk.

[0350] Third, the object at this point was to introduce a new multiplecloning site downstream of the 7.5k promoter in pBS.Vtk.MCS-. The newmultiple cloning site was generated by PCR using 4 different upstreamprimers, and a common downstream primer. Together, these 4 PCR productswould contain either no ATG start codon, or an ATG start codon in eachof the three possible reading frames. In addition, each PCR productcontains at its 3 prime end, translation stop codons in all threereading frames, and a vaccinia virus transcription double stop signal.These 4 PCR products were ligated separately into the NotIl Apal sitesof pBS.Vtk.MCS-, generating the 4 vectors, p7.5/ATGO/tk, p7.5/ATG1I/tk,p7.5/ATG2/tk, and p7.5/ATG3/tk whose sequence modifications relative tothe p7.5/tk vector are shown in FIG. 12. Each vector includes uniqueBamHI, SmaI, PstI, and SalI sites for cloning DNA inserts that employeither their own endogenous translation initiation site (in vectorp7.5/ATGO/tk) or make use of a vector translation initiation site in anyone of the three possible reading frames (p7.5/ATG1/tk, p7.5/ATG3/tk,and p7.5/ATG4/tk).

[0351] In a model experiment cDNA was synthesized from poly-A+mRNA of amurine tumor cell line (BCA39) and ligated into each of the fourmodified p7.5/tk transfer plasmids. The transfer plasmid is amplified bypassage through procaryotic host cells such as E. coli as describedherein or as otherwise known in the art. Twenty micrograms of Not I andApa I digested v/tk vaccinia virus DNA arms and an equimolar mixture ofthe four recombinant plasmid cDNA libraries was transfected into FPVhelper virus infected BSC-1 cells for tri-molecular recombination. Thevirus harvested had a total titer of 6×10⁶ pfu of which greater than 90%were BrdU resistant.

[0352] In order to characterize the size distribution of cDNA inserts inthe recombinant vaccinia library, individual isolated plaques werepicked using a sterile pasteur pipette and transferred to 1.5 ml tubescontaining 100 μl Phosphate Buffered Saline (PBS). Virus was releasedfrom the cells by three cycles of freeze/thaw in dry ice/isopropanol andin a 37° C. water bath. Approximately one third of each virus plaque wasused to infect one well of a 12 well plate containing tk− human 143Bcells in 250 μl final volume. At the end of the two hour infectionperiod each well was overlayed with 1 ml DMEM with 2.5% fetal bovineserum (DMEM-2.5) and with BUdR sufficient to bring the finalconcentration to 125 μg/ml. Cells were incubated in a CO₂ incubator at37° C. for three days. On the third day the cells were harvested,pelleted by centrifugation, and resuspended in 500 μl PBS. Virus wasreleased from the cells by three cycles of freeze/ thaw as describedabove. Twenty percent of each virus stock was used to infect a confluentmonolayer of BSC-1 cells in a 50 mm tissue culture dish in a finalvolume of 3 ml DMEM-2.5. At the end of the two hour infection period thecells were overlayed with 3 ml of DMEM-2.5. Cells were incubated in aCO₂ incubator at 37° C. for three days. On the third day the cells wereharvested, pelleted by centrifugation, and resuspended in 300 μl PBS.Virus was released from the cells by three cycles of freeze/ thaw asdescribed above. One hundred microliters of crude virus stock wastransferred to a 1.5 ml tube, an equal volume of melted 2% low meltingpoint agarose was added, and the virus/agarose mixture was transferredinto a pulsed field gel sample block. When the agar worms weresolidified they were removed from the sample block and cut into threeequal sections. All three sections were transferred to the same 1.5 mltube, and 250 μl of 0.5M EDTA, 1% Sarkosyl, 0.5 mg/ml Proteinase K wasadded. The worms were incubated in this solution at 37° C. for 24 hours.The worms were washed several times in 500 μl 0.5× TBE buffer, and onesection of each worm was transferred to a well of a 1% low melting pointagarose gel. After the worms were added the wells were sealed by addingadditional melted 1% low melting point agarose. This gel was thenelectorphoresed in a Bio-Rad pulsed field gel electrophoresis apparatusat 200 volts, 8 second pulse times, in 0.5× TBE for 16 hours. The gelwas stained in ethidium bromide, and portions of agarose containingvaccinia genomic DNA were excised from the gel and transferred to a 1.5ml tube. Vaccinia DNA was purified from the agarose using β-Agarase(Gibco) following the recommendations of the manufacturer. Purifiedvaccinia DNA was resuspended in 50 μl ddH₂O. One microliter of each DNAstock was used as the template for a Polymerase Chain Reaction (PCR)using vaccinia TK specific primers MM428 and MM430 (which flank the siteof insertion) and Klentaq Polymerase (Clontech) following therecommendations of the manufacturer in a 20 μl final volume. Reactionconditions included an initial denaturation step at 95° C. for 5minutes, followed by 30 cycles of: 94° C. 30 seconds, 55° C. 30 seconds,68° C. 3 minutes. Two and a half microliters of each PCR reaction wasresolved on a 1% agarose gel, and stained with ethidium bromide.Amplified fragments of diverse sizes were observed. When corrected forflanking vector sequences amplified in PCR the inserts range in sizebetween 300 and 2500 bp.

[0353] Representative expression of gene products in this library wasestablished by demonstrating that the frequency of specific cDNArecombinants in the vaccinia library was indistinguishable from thefrequency with which recombinants of the same cDNA occur in a standardplasmid library. This is illustrated in Table 6 for an IAP sequence thatwas previously shown to be upregulated in murine tumors. Twenty separatepools with an average of either 800 or 200 viral pfu from the vaccinialibrary were amplified by infecting microcultures of 143B tk− cells inthe presence of BDUR. DNA was extracted from each infected culture afterthree days and assayed by PCR with sequence specific primers for thepresence of a previously characterized endogenous retrovirus (IAP,intracistemal A particle) sequence. Poisson analysis of the frequency ofpositive pools indicates a frequency of one IAP recombinant forapproximately every 500 viral pfu (Table 6). Similarly, twenty separatepools with an average of either 1,400 or 275 bacterial cfu from theplasmid library were amplified by transformation of DH5 a bacteria.Plasmid DNA from each pool was assayed for the presence of the same IAPsequence. Poisson analysis of the frequency of positive pools indicatesa frequency of one IAP recombinant for every 450 plasmids (Table 6).TABLE 6 Limiting dilution analysis of IAP sequences in a recombinantaccinia library and a conventional plasmid cDNA library #Wells Positiveby PCR F₀ μ Frequency #PFU/well Vaccinia Library 800 18/20 0.05 2.31/350 200  6/20 0.7 0.36 1/560 #CFU/well Plasmid Library 1400  20/20 0 —— 275  9/20 0.55 0.6 1/450

[0354] Similar analysis was carried out with similar results forrepresentation of an alpha tubulin sequence in the vaccinia library. Thecomparable frequency of arbitrarily chosen sequences in the twolibraries constructed from the same tumor cDNA suggests that althoughconstruction of the Vaccinia library is somewhat more complex and iscertainly less conventional than construction of a plasmid library, itis equally representative of tumor cDNA sequences.

[0355] Discussion

[0356] The above-described tri-molecular recombination strategy yieldsclose to 100% viral recombinants. This is a highly significantimprovement over current methods for generating viral recombinants bytransfection of a plasmid transfer vector into vaccinia virus infectedcells. This latter procedure yields viral recombinants at a frequency ofthe order of only 0.1%. The high yield of viral recombinants intri-molecular recombination makes it possible, for the first time, toefficiently construct genomic or cDNA libraries in a vaccinia virusderived vector. In the first series of experiments a titer of 6×10⁶recombinant virus was obtained following transfection with a mix of 20micrograms of NotI and ApaI digested vaccinia vector arms together withan equimolar concentration of tumor cell cDNA. This technologicaladvance creates the possibility of new and efficient screening andselection strategies for isolation of specific genomic and cDNA clones.

[0357] The tri-molecular recombination method as herein disclosed may beused with other viruses such as mammalian viruses including vaccinia andherpes viruses. Typically, two viral arms which have no homology areproduced. The only way that the viral arms can be linked is by bridgingthrough homologous sequences that flank the insert in a transfer vectorsuch as a plasmid. When the two viral arms and the transfer vector arepresent in the same cell the only infectious virus produced isrecombinant for a DNA insert in the transfer vector.

[0358] Libraries constructed in vaccinia and other mammalian viruses bythe tri-molecular recombination method of the present invention may havesimilar advantages to those described here for vaccinia virus and itsuse in identifying target antigens in the CTL screening system of theinvention. Similar advantages are expected for DNA libraries constructedin vaccinia or other mammalian viruses when carrying out more complexassays in eukaryotic cells. Such assays include but are not limited toscreening for DNA encoding receptors and ligands of eukaryotic cells.

Example 6 Preparation of Transfer Plasmids

[0359] The transfer vectors may be prepared for cloning by known means.A preferred method involves cutting 1-5 micrograms of vector with theappropriate restriction endonucleases (for example SmaI and SalI orBamHI and SalI) in the appropriate buffers, at the appropriatetemperatures for at least 2 hours. Linear digested vector is isolated byelectrophoresis of the digested vector through a 0.8% agarose gel. Thelinear plasrnid is excised from the gel and purified from agarose usingmethods that are well known.

[0360] Ligation. The CDNA and digested transfer vector are ligatedtogether using well known methods. In a preferred method 50-100 ng oftransfer vector is ligated with varying concentrations of cDNA using T4DNA Ligase, using the appropriate buffer, at 14° C. for 18 to 24 hours.

[0361] Transformation. Aliquots of the ligation reactions aretransformed by electroporation into E. coli bacteria such as DH10B orDH5 alpha using methods that are well known. The transformationreactions are plated onto LB agar plates containing a selectiveantibiotic (ampicillin) and grown for 14-18 hours at 37° C. All of thetransformed bacteria are pooled together, and plasmid DNA is isolatedusing well known methods.

[0362] Preparation of buffers mentioned in the above description ofpreferred methods according to the present invention will be evident tothose of skill.

Example 7 Introduction of Vaccinia Virus DNA Fragments and TransferPlasmids into Tissue Culture Cells for Trimolecular Recombination

[0363] A cDNA or other library is constructed in the 4 transfer plasmidsas described in Example 5, or by other art-known techniques.Trimolecular recombination is employed to transfer this cDNA libraryinto vaccinia virus. Confluent monolayers of BSC1 cells are infectedwith fowlpox virus HP1 at a moi of 1-1.5. Infection is done in serumfree media supplemented with 0.1% Bovine Serum Albumin. The BSC1 cellsmay be in 12 well or 6 well plates, 60 mm or 100 mm tissue cultureplates, or 25 cm², 75 cm², or 150 cm² flasks. Purified DNA from v7.5/tkor vEL/tk is digested with restriction endonucleases ApaI and NotI.Following these digestions the enzymes are heat inactivated, and thedigested vaccinia arms are purified using a centricon 100 column.Transfection complexes are then formed between the digested vaccinia DNAand the transfer plasmid cDNA library. A preferred method usesLipofectamine or Lipofectamine Plus (Life Technologies, Inc.) to formthese transfection complexes. Transfections in 12 well plates usuallyrequire 0.5 micrograms of digested vaccinia DNA and 10 ng to 200 ng ofplasmid DNA from the library. Transfection into cells in larger culturevessels requires a proportional increase in the amounts of vaccinia DNAand transfer plasmid. Following a two hour infection at 37° C. thefowlpox is removed, and the vaccinia DNA, transfer plasmid transfectioncomplexes are added. The cells are incubated with the transfectioncomplexes for 3 to 5 hours, after which the transfection complexes areremoved and replaced with 1 ml DMEM supplemented with 2.5% Fetal BovineSerum. Cells are incubated in a CO₂ incubated at 37° C. for 3 days.After 3 days the cells are harvested, and virus is released by threecycles of freeze/thaw in dry ice isopropanol/37° C. water bath.

Example 8 Transfection of Mammalian Cells

[0364] This example describes alternative methods to transfect cellswith vaccinia DNA and transfer plasmid. Trimolecular recombination canbe performed by transfection of digested vaccinia DNA and transferplasmid into host cells using for example, calcium-phosphateprecipitation (F. L. Graham, A. J. Van derEb (1973) Virology 52:456-467,C. Chen, H. Okayama (1987) Mol. Cell. Biol. 7:2745-2752), DEAE-Dextran(D. J. Sussman, G. Milman (1984) Mol. Cell. Biol. 4: 1641-1643), orelectroporation (T. K. Wong, E. Neumann (1982) Biochem. Biophys. Res.Commun. 107: 584-587, E. Neumann, M. Schafer-Ridder, Y. Wang, P. H.Hofschneider (1982) EMBO J. 1: 841-845).

Example 9 Construction of MVA Trimolecular Recombination Vectors

[0365] In order to construct a Modified Vaccinia Ankara (MVA) vectorsuitable for trimolecular recombination, two unique restrictionendonuclease sites must be inserted into the MVA tk gene. The completeMVA genome sequence is known (GenBank U94848). A search of this sequencerevealed that restriction endonucleases AscI, RsrII, SfiI, and XmaI donot cut the MVA genome. Restriction endonucleases AscI and XmaI havebeen selected due to the commercial availability of the enzymes, and thesize of the recognition sequences, 8 bp and 6 bp for AscI and XmaIrespectively. In order to introduce these sites into the MVA tk gene aconstruct will be made that contains a reporter gene (E. coli gusA)flanked by XmaI and AscI sites. The Gus gene is available in pCRII.Gus(M. Merchlinsky, D. Eckert, E. Smith, M. Zauderer. 1997 Virology238:444-451). This reporter gene construct will be cloned into atransfer plasmid containing vaccinia tk DNA flanks and the early/late7.5 k promoter to control expression of the reporter gene. The Gus genewill be PCR amplified from this construct using Gus specific primers.Gus sense 5′ ATGTTACGTCCTGTAGAAACC 3′ (SEQ ID NO:94), and Gus Antisense5′TCATTGTTTGCCTCCCTGCTG 3′(SEQ ID NO:95). The Gus PCR product will thenbe PCR amplified with Gus specific primers that have been modified toinclude NotI and XmaI sites on the sense primer, and AscI and ApaI siteson the antisense primer. The sequence of these primers is: NX-Gus Sense5′ AAAGCGGCCGCCCCGGGATGTTACGTCC 3′ (SEQ ID NO:96); and AA-Gus antisense5′ AAAGGGCCCGGCGCGCCTCATTGTTTGCC 3′ (SEQ ID NO:97).

[0366] This PCR product will be digested with NotI and ApaI and clonedinto the NotI and ApaI sites of p7.5/tk (M. Merchlinsky, D. Eckert, E.Smith, M. Zauderer. 1997 Virology 238:444-451). The 7.5 k-XmaI-gusA-AscIconstruct will be introduced into MVA by conventional homologousrecombination in permissive QT35 or BHK cells. Recombinant plaques willbe selected by staining with the Gus substrate X-Glu (5-bromo-3indoyl-β-D-glucuronic acid; Clontech) (M. W. Carroll, B. Moss. 1995Biotechniques 19:352-355). MVA-Gus clones, which will also contain theunique XmaI and AscI sites, will be plaque purified to homogeneity.Large scale cultures of MVA-Gus will be amplified on BHK cells, andnaked DNA will be isolated from purified virus. After digestion withXmaI and AscI the MVA-Gus DNA can be used for trimolecular recombinationin order to construct cDNA expression libraries in MVA.

[0367] MVA is unable to complete its life cycle in most mammalian cells.This attenuation can result in a prolonged period of high levels ofexpression of recombinant cDNAs, but viable MVA cannot be recovered frominfected cells. The inability to recover viable MVA from selected cellswould prevent the repeated cycles of selection required to isolatefunctional cDNA recombinants of interest. A solution to this problem isto infect MVA infected cells with a helper virus that can complement thehost range defects of MVA. This helper virus can provide the geneproduct(s) which MVA lacks that are essential for completion of its lifecycle. It is unlikely that another host range restricted helper virus,such as fowlpox, would be able to complement the MVA defect(s), as theseviruses are also restricted in mammalian cells. Wild type strains ofvaccinia virus would be able to complement MVA. In this case however,production of replication competent vaccinia virus would complicateadditional cycles of selection and isolation of recombinant MVA clones.A conditionally defective vaccinia virus could be used which couldprovide the helper function needed to recover viable MVA from mammaliancells under nonpermissive conditions, without the generation ofreplication competent virus. The vaccinia D4R open reading frame (orf)encodes a uracil DNA glycosylase enzyme. This enzyme is essential forvaccinia virus replication, is expressed early after infection (beforeDNA replication), and disruption of this gene is lethal to vaccinia. Ithas been demonstrated that a stably transfected mammalian cell lineexpressing the vaccinia D4R gene was able to complement a D4R deficientvaccinia virus (G. W. Holzer, F. G. Falkner. 1997 J. Virology71:4997-5002). A D4R deficient vaccinia virus would be an excellentcandidate as a helper virus to complement MVA in mammalian cells.

[0368] In order to construct a D4R complementing cell line the D4R orfwill be cloned from vaccinia strain v7.5/tk by PCR amplification usingprimers D4R-Sense 5′ AAAGGATCCA TAATGAATTC AGTGACTGTA TCACACG 3′ (SEQ IDNO:98), and D4R Antisense 5′ CTTGCGGCCG CTTAATAAAT AAACCCTTGA GCCC3′(SEQ ID NO:99). The sense primer has been modified to include a BamHIsite, and the anti-sense primer has been modified to include a NotIsite. Following PCR amplification and digestion with BamHI and NotI theD4R orf will be cloned into the BamHI and NotI sites of pIRESHyg(Clontech). This mammalian expression vector contains the strong CMVImmediate Early promoter/Enhancer and the ECMV internal ribosome entrysite (IRES). The D4RIRESHyg construct will be transfected into BSC1cells and transfected clones will be selected with hygromycin. The IRESallows for efficient translation of a polycistronic MRNA that containsthe D4Rorf at the 5′ end, and the Hygromycin phosphotransferase gene atthe 3′ end. This results in a high frequency of Hygromycin resistantclones being functional (the clones express D4R). BSC1 cells thatexpress D4R (BSC1.D4R) will be able to complement D4R deficientvaccinia, allowing for generation and propagation of this defectivestrain.

[0369] To construct D4R deficient vaccinia, the D4R orf (position 100732to 101388 in vaccinia genome) and 983 bp (5′ end) and 610 bp (3′end) offlanking sequence will be PCR amplified from the vaccinia genome.Primers D4R Flank sense 5′ ATTGAGCTCT TAATACTTTT GTCGGGTAAC AGAG 3′ (SEQID NO:100), and D4R Flank antisense 5′ TTACTCGAGA GTGTCGCAAT TTGGATTTT3′ (SEQ ID NO:101) contain a Sac (Sense) and XhoI (Antisense) site forcloning and will amplify position 99749 to 101998 of the vacciniagenome. This PCR product will be cloned into the SacI and XhoI sites ofpBluescript II KS (Stratagene), generating pBS.D4R.Flank. The D4R genecontains a unique EcoRI site beginning at nucleotide position 3 of the657bp orf, and a unique PstI site beginning at nucleotide position 433of the orf. Insertion of a Gus expression cassette into the EcoRI andPstI sites of D4R will remove most of the D4R coding sequence. A 7.5 kpromoter- Gus expression vector has been constructed (M. Merchlinsky, D.Eckert, E. Smith, M. Zauderer. 1997 Virology 238:444-451). The 7.5-Gusexpression cassette will be isolated from this vector by PCR usingprimers 7.5 Gus Sense 5′ AAAGAATTCC TTTATTGTCA TCGGCCAAA 3′ (SEQ IDNO:102) and 7.5Gus antisense 5′ AATCTGCAGT CATTGTTTGC CTCCCTGCTG 3′ (SEQID NO:103). The 7.5Gus sense primer contains an EcoRI site and the7.5Gus antisense primer contains a PstI site. Following PCRamplification the 7.5Gus molecule will be digested with EcoRI and PstIand inserted into the EcoRI and PstI sites in pBS.D4R.Flank, generatingpBS.D4R⁻/7.5Gus⁺. D4R⁻/Gus⁺ vaccinia can be generated by conventionalhomologous recombination by transfecting the pBS.D4R⁻/7.5Gus⁺ constructinto v7.5/tk infected BSC1.D4R cells. D4R-/Gus+virus can be isolated byplaque purification on BSC1.D4R cells and staining with X-Glu. The D4R-virus can be used to complement and rescue the MVA genome in mammaliancells.

[0370] In a related embodiment, the MVA genome may be rescued inmammalian cells with other defective poxviruses, and also by apsoralen/UV-inactivated wild-type poxviruses. Psoralen/UV inactivationis discussed herein.

Example 10 Construction and Use of D4R Trimolecular RecombinationVectors

[0371] Poxvirus infection can have a dramatic inhibitory effect on hostcell protein and RNA synthesis. These effects on host gene expressioncould, under some conditions, interfere with the selection of specificpoxvirus recombinants that have a defined physiological effect on thehost cell. Some strains of vaccinia virus that are deficient in anessential early gene have been shown to have greatly reduced inhibitoryeffects on host cell protein synthesis. Production of recombinant cDNAlibraries in a poxvirus vector that is deficient in an early genefunction could, therefore, be advantageous for selection of certainrecombinants that depend on continued active expression of some hostgenes for their physiological effect. Disruption of essential viralgenes prevents propagation of the mutant strain. Replication defectivestrains of vaccinia could, however, be rescued by providing the missingfunction through transcomplementation in host cells or by helper virusthat can be induced to express this gene.

[0372] Infection of a cell population with a poxvirus libraryconstructed in a replication deficient strain should greatly attenuatethe effects of infection on host cell signal transduction mechanisms,differentiation pathways, and transcriptional regulation. An additionaland important benefit of this strategy is that expression of theessential gene under the control of a targeted transcriptionalregulatory region can itself be the means of selecting recombinant virusthat directly or indirectly lead to activation of that transcriptionalregulatory region. Examples include the promoter of a gene activated asa result of crosslinking surface immunoglobulin receptors on early Bcell precursors or the promoter of a gene that encodes a marker inducedfollowing stem cell differentiation. If such a promoter drivesexpression of an essential viral gene, then only those viralrecombinants that directly or indirectly activate expression of thattranscriptional regulator will replicate and be packaged as infectiousparticles. This method has the potential to give rise to much lowerbackground then selection methods based on expression of dipA or a CTLtarget epitope because uninduced cells will contain no replicationcompetent vaccinia virus that might be released through non-specificbystander effects. The selected recombinants can be further expanded ina complementing cell line or in the presence of a complementing helpervirus or transfected plasmid.

[0373] A number of essential early vaccinia genes have been described.Preferably, a vaccinia strain deficient for the D4R gene could beemployed. The vaccinia D4R open reading frame (orf) encodes a uracil DNAglycosylase enzyme. This enzyme is reqired for viral DNA replication anddisruption of this gene is lethal to vaccinia (A. K. Millns, M. S.Carpenter, and A. M. Delange. 1994 Virology 198:504-513). It has beendemonstrated that a stably transfected mammalian cell line expressingthe vaccinia D4R gene is able to complement a D4R deficient vacciniavirus (G. W. Holzer, F. G. Falkner. 1997 J. Virology 71: 4997-5002). Inthe absence of D4R complementation, infection with the D4R deficientvaccinia results in greatly reduced inhibition of host cell proteinsynthesis (Holzer and Falkner). It has also been shown that a foreigngene inserted into the tk gene of D4R deficient vaccinia continues to beexpressed at high levels, even in the absence of D4R complementation (M.Himly, M. Pfleiderer, G. Holzer, U. Fischer, E. Hannak, F. G. Falkner,and F. Dorner. 1998 Protein Expression and Purification 14: 317-326).The replication deficient D4R strain is, therefore, well-suited forselection of viral recombinants that depend on continued activeexpression of some host genes for their physiological effect.

[0374] To implement this strategy for selection of specific recombinantsfrom representative cDNA libraries constructed in a D4R deficientvaccinia strain the following cell lines and vectors are required:

[0375] 1. D4R expressing complementing cell line is required forexpansion of D4R deficient viral stocks.

[0376] 2. The D4R gene must be deleted or inactivated in a viral strainsuitable for trimolecular recombination.

[0377] 3. Plasmid or viral constructs must be generated that express D4Runder the control of different inducible promoters such as that whichregulates expression of BAX or other genes induced followingcrosslinking of membrane immunoglobulin receptors on CH33 B lymphomacells or the promoter for expression of type X collagen followinginduction of chondrocyte differentiation from C3H10T1/2 progenitorcells. Stable transfectants of these constructs in the relevant cellline are required to rescue specific recombinants. Alternatively, ahelper virus expressing the relevant construct can be employed forinducible expression in either cell lines or primary cultures.

[0378] 10.1 Construction of a D4R Complementing Cell Line A D4Rcomplementing cell line is constructed as follows. First, the D4R orf(position 100732 to 101388 in vaccinia genome) is cloned from vacciniastrain v7.5/tk by PCR amplification using the following primers:D4R-sense, 5′ AAAGAATTCA TAATGAATTC AGTGACTGTA TCACACG 3′, designatedherein as SEQ ID NO:104; and D4R-antisense: 5′ CTTGGATCCT TAATAAATAAACCCTTGAGC CC 3′, designated herein as SEQ ID NO:105.

[0379] The sense primer is modified to include an EcoRI site, and theanti-sense primer is modified to include a BamHI site (both underlined).Following standard PCR amplification and digestion with EcoRI and BamHI,the resulting D4R orf is cloned into the EcoRI and BamHI sites ofpIRESneo (available from Clontech, Palo Alto, Calif.). This mammalianexpression vector contains the strong CMV immediate earlypromoter/enhancer and the ECMV internal ribosome entry site (IRES). TheD4R/IRESneo construct is transfected into BSC1 cells and transfectedclones are selected with G418. The IRES allows for efficient translationof a polycistronic mRNA that contains the D4Rorf at the 5′ end, and theneomycin phosphotransferase gene at the 3′ end. This results in a highfrequency of G418 resistant clones being functional (the clones expressD4R). Transfected clones are tested by northern blot analysis using theD4R gene as probe in order to identify clones that express high levelsof D4R MRNA. BSC1 cells that express D4R (BSC1.D4R) are able tocomplement D4R deficient vaccinia, allowing for generation andpropagation of D4R defective viruses.

[0380] 10.2. Construction of D4R Deficient vaccinia vector AD4R-deficient vaccinia virus, suitable for trimolecular recombination asdescribed in Example 5, supra, is constructed by disruption of the D4Rorf (position 100732 to 101388 in vaccinia genome) through the insertionof an E. Coli GusA expression cassette into a 300-bp deletion, by thefollowing method.

[0381] In order to insert the GusA gene, regions flanking the insertionsite are amplified from vaccinia virus as follows. The left flankingregion is amplified with the following primers: D4R left flank sense:5′ AATAAGCTTT ACTCCAGATA ATATGGA 3′, designated herein as SEQ ID NO:106;and D4R left flank antisense: 5′ AATCTGCAGC CCAGTTCCAT TTT 3′,designated herein as SEQ ID NO:107.

[0382] These primers amplify a region extending from position 100167 toposition 100960 of the vaccinia genome, and have been modified toinclude a HindIII (Sense) andPstI (Antisense) site for cloning (bothunderlined). The resulting PCR product is digested with HindIII andPstI, and cloned into the HindIII and PstI sites of pBS (available fromStratagene), generating pBS.D4R.LF. The right flanking region isamplified with the following primers: D4R right flank sense:5′ AATGGATCCT CATCCAGCGG CTA 3′, designated herein as SEQ ID NO:108; andD4R right flank antisense: 5′ AATGAGCTCT AGTACCTACA ACCCGAA 3′,designated herein as SEQ ID NO:109.

[0383] These primers amplify a region extending from position 101271 toposition 101975 of the vaccinia genome, and have been modified toinclude a BamHi (Sense) and SacI (Antisense) site for cloning (bothunderlined). The resulting PCR product is digested with BamHI and SacI,and cloned into the BamHI and SacI sites of pBS.D4R.LF, creatingpBS.D4R.LF/RF.

[0384] An expression cassette comprising the GusA coding region operablyassociated with a poxvirus synthetic early/late (E/L) promoter, isinserted into pBS.D4R.LF/RF by the following method. The E/L promoter-Gus cassette is derived from the pEL/tk-Gus construct described inMerchlinsky, M., et al., Virology 238: 444-451 (1997). The NotI siteimmediately upstream of the Gus ATG start codon is removed by digestionof pEL/tk-Gus with NotI, followed by a fill in reaction with Klenowfragment and religation to itself, creating pEL/tk-Gus(NotI-). TheE/L-Gus expression cassette is isolated from pEL/tk-Gus(NotI−) bystandard PCR using the following primers: EL-Gus sense: 5′ AAAGTCGACGGCCAAAAATT GAAATTTt 3′, designated herein as SEQ ID NO:110; and EL-Gusantisense: 5′ AATGGATCCT CATTGTTTGC CTCCC 3′, designated herein as SEQID NO:111.

[0385] The EL-Gus sense primer contains a SalI site and the EL-Gusantisense primer contains a BamHI site (both underlined). Following PCRamplification the EL-Gus cassette is digested with SalI and BamHI andinserted into the SalI and BamHI sites in pBS.D4R.LF/RF generatingpBS.D4R⁻/ELGus. This transfer plasmid contains an EL-Gus expressioncassette flanked on both sides by D4R sequence. There is also a 300 bpdeletion engineered into the D4R orf.

[0386] D4R⁻/Gus⁺ vaccinia viruses suitable for trimolecularrecombination are generated by conventional homologous recombinationfollowing transfection of the pBS.D4R⁻/ELGus construct intov7.5/tk-infected BSC1.D4R cells. D4R−/Gus⁺ virus are isolated by plaquepurification on BSC1.D4R cells and staining with X-Glu (M. W. Carroll,B. Moss. 1995. Biotechniques 19: 352-355). This new strain is designatedv7.5/tk/Gus/D4R.

[0387] DNA purified from v7.5/tk/Gus/D4R is used to constructrepresentative vaccinia cDNA libraries by trimolecular recombinationaccording to the method described in Example 5, except that thereactions are carried out in the BSC1.D4R complementing cell line.

[0388] 10.3. Preparation of host cells expressing D4R under the controlof inducible promoters Host cells which express the D4R gene uponinduction of an inducible promoter are prepared as follows. Plasmidconstructs are generated that express the vaccinia D4R gene under thecontrol of an inducible promoter. Examples of inducible promotersinclude, but are not limited to, promoters which are upregulatedfollowing crosslinking of membrane immunoglobulin on CH33 cells (forantibody selection), e.g., the BAX promoter as described in Examples 2and 3. The vaccinia D4R orf is amplified by PCR using primers D4R senseand D4R antisense described above in section 10.1. These PCR primers aremodified as needed to include desirable restriction endonuclease sites.The D4R orf is then cloned in a suitable eukaryotic expression vector(which allows for the selection of stably transformed cells) in operableassociation of any desired promoter employing methods known to thoseskilled in the art.

[0389] The construct is then transfected into a suitable host cell, forexample, the for selection of antibodies as described in Examples 2 and3, the D4R gene, in operable association with the BAX promoter, isstably transfected into a suitable cell line, for example, a CH33 cellline, a CH 31 cell line, or a WEM-231 cell line. The resulting hostcells are utilized in the production of antibodies, essentially asdescribed in Example 3, using libraries prepared in v7.5/tk/Gus/D4R.Antigen-induced cross-linking of membrane-expressed immunoglobulinmolecules on the surface of host cells results in the induction ofexpression of the D4R gene product in the cross-linked cells. Expressionof D4R complements the defect in the v7.5/tk/Gus/D4R genomes in whichthe libraries are produced, allowing the production of infectious virusparticles.

Example 11 Attenuation of Poxvirus Mediated Host Shut-off by ReversibleInhibitor of DNA Synthesis

[0390] As discussed infra, attenuated or defective virus is sometimesdesired to reduce cytopathic effects. Cytopathic effects during viralinfection might interfere with selection and identification ofimmunoglobulin molecules using methods which take advantage of host celldeath (e.g. apoptosis induced by cross-linking). Such effects can beattenuated with a reversible inhibitor of DNA synthesis such ashydroxyurea (HU) (Pogo, B. G. and S. Dales Virology, 1971.43(1):144-51). ffU inhibits both cell and viral DNA synthesis bydepriving replication complexes of deoxyribonucleotide precursors(Hendricks, S. P. and C. K. Mathews J Biol Chem, 1998.273(45):29519-23). Inhibition of viral DNA replication blocks late viralRNA transcription while allowing transcription and translation of genesunder the control of early vaccinia promoters (Nagaya, A., B. G. Pogo,and S. Dales Virology, 1970. 40(4):1039-51). Thus, treatment withreversible inhibitor of DNA synthesis such as HU allows the detection ofeffects of cross-linking. Following appropriate incubation, HUinhibition can be reversed by washing the host cells so that the viralreplication cycle continues and infectious recombinants can be recovered(Pogo, B.G. and S. Dales Virology, 1971. 43(1):144-51).

[0391] The results in FIG. 9 demonstrate that induction of type Xcollagen synthesis, a marker of chondrocyte differentiation, in C3H10T ½progenitor cells treated with BMP-2 (Bone Morphogenetic Protein-2) isblocked by vaccinia infection but that its synthesis can be rescued byHU mediated inhibition of viral DNA synthesis. When HU is removed fromcultures by washing with fresh medium, viral DNA synthesis and assemblyof infectious particles proceeds rapidly so that infectious viralparticles can be isolated as soon as 2 hrs post-wash.

[0392] C3H10T ½ cells were infected with WR vaccinia virus at MOI=1 and1 hour later either medium or 400 ng/ml of BMP-2 in the presence orabsence of 2 mM HU was added. After a further 21 hour incubation at 37°C., HU was removed by washing with fresh medium. The infectious cyclewas allowed to continue for another 2 hours to allow for initiation ofviral DNA replication and assembly of infectious particles. At 24 hoursRNA was extracted from cells maintained under the 4 different cultureconditions. Northern analysis was carried out using a type X collagenspecific probe. The uninduced C3H10T½ cells have a mesenchymalprogenitor cell phenotype and as such do not express type X collagen(first lane from left). Addition of BMP-2 to normal, uninfected C3H10T ½cells induces differentiation into mature chondrocytes and expression oftype X collagen (compare first and second lanes from left), whereasaddition of BMP-2 to vaccinia infected C3H10T ½ cells fails to inducesynthesis of type X collagen (third lane from left). In the presence of2 mM HU, BMP-2 induces type X collagen synthesis even in vaccinia virusinfected C3H10T ½ cells (fourth lane from left).

[0393] This strategy for attenuating viral cytopathic effects isapplicable to other viruses, other cell types and to selection ofimmunoglobulin molecules that, for example, induce apoptosis uponcross-linking.

Example 12 Construction of Human Fab Fragment Libraries of DiverseSpecificity

[0394] Libraries of polynucleotides encoding fully human, diverseimmunglobulin Fab fragments are produced as follows. These Fab fragmentscomprise a heavy chain variable region linked to a first constant regiondomain (VH-CH1) paired with an immunoglobulin light chain. Genes forhuman VH (variable region of heavy chain), VK (variable region of kappalight chain) and VL (variable region of lambda light chains) areamplified by PCR. For each of the three variable gene families, both arecombinant plasmid library and a vaccinia virus library is constructed.The variable region genes are inserted into a p7.5/tk-basedtransfer/expression plasmid immediately upstream of a constant regionsequence corresponding to the CH1 domain of heavy chains or the kappalight chain constant region, CK. These plasmids are employed to generatethe corresponding vaccinia virus recombinants by trimolecularrecombination and can also be used directly for high level expression ofFab fragments following transfection of one immnunoglobulin chain orfragment thereof into cells infected with vaccinia virus recombinants ofa second immunoglobulin chain or fragment thereof. The two chains aresynthesized and assembled to form an Fab fragment. These Fab fragmentsmay be membrane bound or secreted by attaching coding sequences forsignal sequences, transmembrane domains, and/or intracellular domains,as is undertood by one of ordinary skill in the art.

[0395] 12.1 pVHEc. An expression vector which encodes a human heavychain fragment comprising VH and the CHI domain of Cμ, designated pVHEc,is constructed as follows. Plasmid p7.5/tk2 is produced as described inExample 1.1, supra. A DNA construct encoding amino acids 109-113 of VHand the CH1 domain, i.e., amino acids 109-223B of Cμ, is amplified fromthe IgM heavy chain gene isolated as described in Example 1, and ismodified by PCR to include a BstEII site at the 5′ end of the regionencoding amino acids 109-113+ the Cμ CH1 domain, and a stop codon and aSalI site at its 3′ end. This DNA is inserted into p7.5/tk2 between theBstEII and SalI sites to generate pVBEc. Heavy chain variable region(VH) PCR products (amino acids (−4) to (110)), produced as described inExample 1.4(a), using the primers listed in Tables 1 and 2, are clonedinto BssHII and BstEII sites. Because of the overlap between the CH1domain sequence and the restriction enzyme sites selected, this resultsin construction of a contiguous heavy chain fragment which lacks afunctional signal peptide but remains in the correct translationalreading frame.

[0396] 12.2 pVKEc and pVLEc. Expression vectors encoding the human κ andλ immunoglobulin light chain constant regions, designated herein aspVKEc and pVLEc, are constructed as follows. Plasmid p7.5/tk3.1, isproduced as described in Example 1.3, supra.

[0397] (a) Plasmid p7.5/tk3.1 is converted into pVKEc by the followingmethod. A cDNA coding for the C_(κ) region is isolated as described inExample 1, with primers to include an XhoI site at the 5′ end of theregion encoding amino acids 104-107+C_(κ), and a stop codon and a SalIsite at its 3′ end, which is then cloned into p7.5/tk3.1 at XhoI andSalI sites to generate pVKEc. Kappa light chain variable region (VK) PCRproducts (amino acids(−3) to(105)), produced as described in Example1.4(b), using the primers listed in Tables 1 and 2, are then cloned intopVKEc at the ApaLI and XhoI sites. Because of the overlap between the κlight chain sequence and the restriction enzyme sites selected, thisresults in construction of contiguous κ light chains which lacks afunctional signal peptide but remains in the correct translationalreading frame.

[0398] (b) Plasmid p7.5/tk3.1 is converted into pVLEc by the followingmethod. A cDNA coding for the C_(κ) region is isolated as described inExample 1, with primers to include a HindIII site and amino acids 105 to107 of V_(λ) at its 5′ end and a stop codon and a SalI site at its 3′end, which is then cloned into p7.5/tk3 at HindIII and SalI sites togenerate pVLEc. Lambda light chain variable region (VL) PCR products(amino acids (−3) to(104)), produced as described in Example 1.4(c),using the primers listed in Tables 1 and 2, are then cloned into pVLEcat ApaLI and HindIII sites. Because of the overlap between the λ lightchain sequence and the restriction enzyme sites selected, this resultsin construction of contiguous λ light chains which lacks a functionalsignal peptide but remains in the correct translational reading frame.

[0399] 12.3 Secreted orMembrane BoundForms of Fab. The expressionvectors (pVHEc, pVKEc and pVLEc) serve as prototype vectors into whichsecretion signals, transmembrane domains, cytoplasmic domains, orcombinations thereof can be cloned to target Fab to the cell surface orthe extracellular space. These signals and domains, examples of whichare shown in Table 7, may be inserted either in the N-terminus of Fabbetween NcoI and BssHII of pHEc (or NcoI and ApaLI of pVKEc and pVLEc)and/or in the C-terminus at SalI site. To target an Fab for secretioninto the extracellular compartment, a signal peptide is inserted at theN-terminus of either or both Fab chains, VH-CH1 or light chain. Toanchor an Fab in the plasma membrane for extracellular presentation, atransmembrane domain is added to the carboxyl-terninus of VH-CH1 chainand/or to the light chain. A cytoplasmic domain may also be added. TABLE7 Localization signals Signal sequence Terminus Location ProteinMGWSCIILFLVATATGAHS N ES IgG1 (SEQ ID NO:146) NLWTTASTFIVLFLLSLFYSTTVTLFC/N PM IgM (SEQ ID NO:147)

[0400] Abbreviations for items under Location: ES, extracellular space;PM, plasma membrane.

Example 13 Construction of Human Single-Chain-Fv (ScFv) AntibodyLibraries

[0401] 13.1 Human scFv expression vectors p7.5/tk3.2 and p7.5/tk3.3 areconstructed by the following method, as illustrated in FIG. 10. Plasmidp7.5/tk3 is produced as described in Example 1.3, supra. Plasmidp7.5/tk3 is converted to p7.5/tk3.1 by changing the four nucleotidesATAC between NcoI and ApaLI sites into ATAGC, so that the ATG startcodon in NcoI is in-frame with ApaLI without the inserted signalpeptide. This is conveniently accomplished by replacing the NotI-to-SalIcassette described in Example 1.3 (SEQ ID NO:29) with a cassette havingthe sequence 5′-GCGGCCGCCC ATGGATAGCG TGCACTTGAC TCGAGAAGCT TAGTAGTCGAC-3′, referred to herein as SEQ ID NO:112.

[0402] Plasmid p7.5/tk3.1 is converted to p7.5/tk3.2 by substituting theregion between XhoI and SalI (i.e., nucleotides 30 to 51 of SEQ IDNO:112), referred to herein as SEQ ID NO:113, with the followingcassette: XhoI-(nucleotides encoding amino acids 106-107 ofVκ)-(nucleotides encoding a 10 amino acidlinker)-G-BssHII-ATGC-BstEII-(nucleotides encoding amino acids 111-113of VH)-stop codon-SalI. This is accomplished by digesting p7.5/tk3.1with XhoI and SalI, and inserting a cassette having the sequence5′CTCGAGAT CAAAGAGGGT AAATCTTCCG GATCTGGTTC CGAAGGCGCG CATGCGGTCACCGTCTCCTC ATGAGTCGAC 3′, referred to herein as SEQ ID NO:114. Thelinker between Vκ and VH will have a final size of 14 amino acids, withthe last 4 amino acids contributed by the VH PCR products, inserted asdescribed below. The sequence of the linker is 5′GAG GGT AAA TCT TCC GGATCT GGT TCC GAA GGC GCG CAC TCC 3′ (SEQ ID NO: 115), which encodes aminoacids EGKSSGSGSEGAHS (SEQ ID NO:116).

[0403] Plasmid p7.5/tk3.1 is converted to p7.5/tk3.3 by substituting theregion between HindIII and SalI (i.e., nucleotide 36 to 51 of SEQ IDNO:112), referred to herein as SEQ ID NO:117, with the followingcassette: HindIII-(nucleotides encoding amino acid residues 105-107 ofVλ)-(nucleotides encoding a 10 amino acidlinker)-G-BssHII-ATGC-BstEII-(nucleotides encoding amino acids 111-113of VH)-stop codon-SalI. This is accomplished by digesting p7.5/tk3.1with HindIII and SalI, and inserting a cassette having the sequence 5 ′AAGCTTACCG TCCTAGAGGG TAAATCTTCC GGATCTGGTTC CGAAGGCGCG CATGCGGTCACCGTCTCCTC ATGAGTCGAC 3′ (SEQ ID NO:118). The linker between Vλ and VHwill have a final size of 14 amino acids, with the last 4 amino acidscontributed by the VH PCR products, inserted as described below. Thesequence of the linker is 5′GAG GGT AAA TCT TCC GGA TCT GGT TCC GAA GGCGCG CAC TCC 3′ (SEQ ID NO:119), which encodes amino acids EGKSSGSGSEGAHS(SEQ ID NO:120).

[0404] 13.2 Cytosolic Forms of scFv. Expression vectors encoding scFvpolypeptides comprising human κ or λ immunoglobulin light chain variableregions, fused in frame with human heavy chain variable regions, areconstructed as follows.

[0405] (a) Cytosolic VκVH scFv expression products are prepared asfollows. Kappa light chain variable region (Vκ) PCR products (aminoacids (−3) to(105)), produced as described in Example 1.4(b), using theprimers listed in Tables 1 and 2, are cloned into p7.5/tk3.2 between theApaLI and XhoI sites. Because of the overlap between the κ light chainsequence and the restriction enzyme sites selected, this results inconstruction of a contiguous κ light chain in the same translationalreading frame as the downstream linker. Heavy chain variable region (VH)PCR products (amino acids (-4) to(110)), produced as described inExample 1.4(a), using the primers listed in Tables 1 and 2, are clonedbetween the BssHIII and BstEII sites of p7.5/tk3.2 to form complete scFvopen reading frames. The resulting products are cytosolic forms of Vκ-VHfusion proteins connected by a linker of 14 amino acids. The scFv isalso preceded by 6 extra amino acids at the amino terminus encoded bythe restriction sites and part of the Vκ signal peptide.

[0406] (b) Cytosolic VλVH scFv expression products are prepared asfollows. Lambda light chain variable region (VL) PCR products (aminoacids(−3) to (104)), produced as described in Example 1.4(c), using theprimers listed in Tables 1 and 2, are cloned into p7.5/tk3.3 between theApaLI and HindM sites. Because of the overlap between the λ light chainsequence and the restriction enzyme sites selected, this results inconstruction of a contiguous λ light chain in the same translationalreading frame as the downstream linker. Heavy chain variable region (VH)PCR products (amino acids (−4) to (110)), produced as described inExample 1.4(a), using the primers listed in Tables 1 and 2, are clonedbetween BssHII and BstEII sites of p7.5/tk3.3 to form complete scFv openreading frames. The resulting products are cytosolic forms of Vλ-VHfusion proteins connected by a linker of 14 amino acids. The scFv isalso preceded by 6 extra amino acids at the amino terminus encoded bythe restriction sites and part of the Vλ signal peptide.

[0407] 13.3 Secreted or Membrane Bound Forms of scFv. The cytosolic scFvexpression vectors described in section 13.2 serve as the prototypevectors into which secretion signals, transmembrane domains, cytoplasmicdomains, or combinations thereof can be cloned to target scFvpolypeptides to the cell surface or the extracellular space. Examples ofsignal peptides and membrane anchoring domains are shown in Table 7,supra. To generate scFv polypeptides to be secreted into theextracellular space, a cassette encoding an in-frame secretory signalpeptide is inserted so as to be expressed in the N-terminus of scFvpolypeptides between the NcoI and ApaLI sites of p7.5/tk3.2 orp7.5/tk3.3. To generate membrane-bound scFv for Ig-crosslinkingorig-binding based selection, in addition to the signal peptide, acassette encoding the membrane-bound form of Cμ is cloned into theC-terminus of scFv between the BstEII and SalI sites, downstream of andin-frame with the nucleotides encoding amino acids 111-113 of VH. Acytoplasmic domain may also be added.

Example 14 Construction of Camelized Human Single-Domain AntibodyLibraries

[0408] Camelid species use only heavy chains to generate antibodies,which are termed heavy chain antibodies. The poxvirus expression systemis amendable to generate both secreted and membrane-bound humansingle-domain libraries, wherein the human V_(H) domain is “camelized,”i.e., is altered to resemble the V_(H)H domain of a camelid antibody,which can then be selected based on either functional assays orIg-crosslinking/binding. Human V_(H) genes are camelized by standardmutagenesis methods to more closely resemble camelid V_(H)H genes. Forexample, human V_(H)3 genes, produced using the methods described inExample 1.4 using appropriate primer pairs selected from Tables 1 and 2,is camelized by substituting G44 with E, L45 with R, and W47 with G orI. See, e.g., Riechmann, L., and Muyldermans, S. J. Immunol. Meth.231:25-38. To generate a secreted single-domain antibody library,cassettes encoding camelized human VH genes are cloned into pVHEs,produced as described in Example 1.2, to be expressed in-frame betweenthe BssHII and BstEII sites. To generate a membrane-bound single-domainantibody library, cassettes encoding camelized human V_(H) genes arecloned into pVHE, produced as described in Example 1. 1, to be expressedin-frame between the BssHII and BstEII sites. Vectors pVHE and pVHEsalready have the signal peptide cloned in between the NcoI and BssHIIsites. Amino acid residues in the three CDR regions of the camelizedhuman V_(H) genes are subjected to extensive randomization, and theresulting libraries can be selected in poxviruses as described herein.

Example 15 Selection of Fc-Modified Antibodies for Enhanced ImmuneEffector Functions

[0409] Human monoclonal antibodies are being used in therapeuticapplications for treatment of an increasing number of human diseases.Human antibodies may induce or block signaling through specific cellreceptors. In some applications, human antibodies may activate any of avariety of accessory effector cells through an interaction between theFc portion of the antibody molecule and a matching Fc receptor (FcR) onthese effector cells. It is, therefore, of considerable interest toidentify modifications of immunoglobulin heavy chain constant regionsequences that enhance or inhibit binding and signaling through FcR orbinding and activation of other mediators of immune effector functionssuch as components of the complement cascade. See. e.g., U.S. Pat. No.5,624,821; Xu, D., et al., Cell Immunol 200:16-26 (2000); and U.S. Pat.No. 6,194,551, the disclosures of which are incorporated herein byreference in their entireties.

[0410] One such specific effector function is antibody-dependent cellcytotoxicity (ADCC), a process in which antibody-coated target cells aredestroyed by NK cells or other monocytes. ADCC is mediated by antibodymolecules with variable region encoded specificity for a surfacemolecule of a target cell and constant region encoded specificity forFcγRIII on the NK cell. Through analysis of crystal structures andsite-directed mutagenesis, it has been determined that the FcγRIIIbinding site on human IgG1 is localized mainly to the lower hinge, i.e.,about amino acids 247-252 of IgG1, and the adjacent CH2 regions. See,e.g., Sarmay G., et al., Mol Immunol 29:633-639 (1992); and Michaelsen,T. E., et al., Mol Immunol 29:319-26 (1992). By constructing a libraryof genes encoding antibody molecules with randomly mutated lower hingeregions in a selectable mammalian expression vector, it would befeasible to select specific constant region variants with enhancedfunction for ADCC. To simplify execution of this strategy, a library isconstructed with defined immunoglobulin variable region sequences thatconfer a desired specificity.

[0411] 15.1. Construction of pVHE-X and pVKE-X or pVLE-X. PlasmidpVHE-X, a human VH expression vector with a defined variable region,designated herein as X, is constructed as follows. The construction isillustrated in FIG. 11. An antibody with a defined specificity X isisolated by conventional methods, or is produced and selected ineukaryotic cells using poxvirus vectors, by methods described herein. Ifnecessary, the VH gene of the antibody is subcloned into pVHE, producedas described in Example 1.1, between the BssHII/BstEII sites, resultingin plasmid pVHE-X. Also if necessary, the VK or VL gene of the antibodyis subcloned either into pVKE, produced as described in Example 1.3, atthe ApaLI/XhoI sites to produce pVKE-X, or into pVLE, produced asdescribed in Example 1.3, at ApaLI/HindIIIsites, to produce pVLE-X,respectively.

[0412] 15.2 Isolation of a human Cγ1cassette. A cDNA coding for thehuman Cγ1 heavy chain is isolated from bone marrow RNA using SMART™ RACEcDNA Amplification Kit, using the following primers: huCγ1-5B: 5′ATTAGGATCC GGTCACCGTC TCCTCAGCC 3′ (SEQ ID NO:121) huCγ1-3S: 5′ATTAGTCGAC TCATTTACCC GGAGACAGGG AGAG 3′ (SEQ ID NO:122)

[0413] The PCR product comprises the following elements:BamHI-BstEII-(nucleotides encoding amino acids 111-113 ofVH)-(nucleotides encoding amino acids 114-478 of Cγ1)-TGA-SalI. Thisproduct is subcloned into pBluescriptII/KS at BamHI and SalI sites, anda second BstEII site corresponding to amino acids 191 and 192 within theCH1 domain of Cγ1 is removed by site-directed mutagenesis without changeto the amino acid sequence.

[0414] 15.3 Construction of Fcγ1 library. Cγ1 variants are generated byoverlap PCR by the following method. The BstEII-mutagenized Cylcassette,produced as described in section 15.2, is used as the template In thefirst round of PCR, amplifications are carried out in two separatereactions using Cγ1-sense/Cγ1-internal-R and Cγ1-internal-S/Cγ1-reverseprimer sets. Cγ1-sense: 5′ AATATGGTCACCGTCTCCTCAGCC 3′ (SEQ ID NO:123)Cγ1-internal-R: 5′ (MNN)₆TTCAGGTGCTGGGCACGG 3′ (SEQ ID NO:124)Cγ1-internal-S: 5′ (NNK)₆GTCTTCCTCTTCCCCCCA 3′ (SEQ ID NO:125)Cγ1-reverse: 5′ AATATGTCGACTCATTTACCCGG 3′ (SEQ ID NO:126) (M = A + C, K= G + T, N = A + T + G + C)

[0415] The Cγ1-internal-R and Cγ1-intemal-S primers have degeneratesequence tails that code for variants of the six amino acids comprisingresidues 247-252 in the lower hinge. In the second round of PCR, thepurified products from the first round are fused by overlap PCR usingthe Cγ1-sense and Cγ1-reverse primers.

[0416] The resulting products are approximately 1000 bp in size, andrandomly encode all 20 amino acids in each of the six amino acidpositions 247-252. The PCR products are digested with BstEII and SalI,and are cloned into BstEII/SalI-digested pVHE-X, produced as describedin section 15.1, to generate a library of pVHE-X-γ1 variants. Thesevariants are then introduced into vaccinia virus using trimolecularrecombination as described in Example 5. In conjunction with therecombinant vaccinia virus harboring the light chain, the Fcγ1 librarywill be used to select those Fc variants that confer enhanced ADCCactivity on a VHE-X-γ1 expressing antibody.

[0417] 15.4 Other applications. In addition the generation of variantsat amino acids 247-252, other residues, such as amino acids 278-282 andamino acids 346-351of IgG1, are also involved in binding to FcγRIII.Following the identification of Fcγ1 variant in amino acids 247-252 thatexhibits an enhanced ADCC activity, the same strategy can be employed toidentify additional mutations in the other two regions that exhibitsynergistic enhancement of ADCC function.

[0418] The same principle/technique can be applied to identifyingvariants that confer enhanced effector function on other immunoglobulinheavy chain constant region isotypes that bind to different Fcreceptors. In preferred embodiments the receptors to be targeted includeFcγRI (CD64), FcγRII-A (CD32), FcγRII-B1, FcγRII-B2, FcγRIII (CD16), andFcεRI. In other preferred embodiments, variants may be selected thatenhance binding of complement components to the Fc region or Fc mediatedbinding to placental membrane for transplacental transport.

Example 16 Construction of Heavy Chain Fusion Proteins to FacilitateSelection of Cells Infected With Specific Immunoglobulin GeneRecombinant Vaccinia Virus

[0419] 16.1 Construction of CH1-Fas. An expression vector which encodesa fusion protein comprising the human heavy chain CH1 domain of Cμ,fused to the transmembrane and death domains of Fas, designated hereinas CH1-Fas, is constructed by the following method. The fusion proteinis illustrated in FIG. 13(a).

[0420] Plasmid pVHE, produced as described in Example 1.1, is digestedwith BstEII and SalI and the smaller DNA fragment of about 1.4 Kb is gelpurified. This smaller fragment is then used as a template in a PCRreaction using forward primerCH1(F)-5′ACACGGTCAC CGTCTCCTCA GGGAGTGC 3′(SEQ ID NO:127) and reverse primer CH1(R) 5′AGTTAGATCT GGATCCTGGAAGAGGCACGTT 3′ (SEQ ID NO:128). The resulting PCR product of about 320base pairs is gel purified.

[0421] A DNA fragment comprising the transmembrane and death domains ofFas is amplified from plasmid pBS-APO14.2 with forward primer FAS(F)5′AACGTGCCTC TTCCAGGATC CAGATCTAAC 3′ (SEQ ID NO:129) and reverse primerFAS(R) 5′ACGCGTCGAC CTAGACCAAG CTTTGGATTT CAT 3′(SEQ ID NO:130). Theresulting PCR product of about 504 base pairs is gel purified.

[0422] The resulting 320 and 504-base pair fragments are then combinedin a PCR using forward primer CH1(F) and reverse primer FAS (R), toproduce a fusion fragment of about 824 base pairs. This fragment isdigested with BstEII and SalI, and the resulting 810-base pair fragmentis gel purified. Plasmid pVHE also digested with BstEII and SalI, andthe larger resulting fragment of about 5.7 Kb is gel purified. These twoBstEII/SalI fragments are then ligated to produce CH1-Fas.

[0423] 16.2 Construction of CH4-Fas. An expression vector which encodesa fusion protein comprising the human heavy chain CH1-CH4 domains of Cμ,fused to the transmembrane and death domains of Fas, designated hereinas CH4-Fas, is constructed by the following method. The fusion proteinis illustrated in FIG. 13(b).

[0424] Plasmid pVHE, produced as described in Example 1.1, is digestedwith BstEII and SalI and the smaller DNA fragment of about 1.4 Kb is gelpurified. This smaller fragment is then used as a template in a PCRreaction using forward primer CH4(F) 5′ CTCTCCCGCG GACGTCTTCGT 3′ (SEQID NO:131) and reverse primer CH4(R) 5′AGTTAGATCT GGATCCCTCA AAGCCCTCCTC 3′ (SEQ ID NO:132). The resulting PCR product of about 268 base pairsis gel purified.

[0425] A DNA fragment comprising the transmembrane and death domains ofFas is amplified from plasmid pBS-APO14.2 with forward primerFAS(F2)-5′GAGGAGGGCT TTGAGGGATC CAGATCTAAC 3′ (SEQ ID NO:133) andreverse primer FAS(R), as shown in section 16.1. The resulting PCRproduct of about 504 base pairs is gel purified.

[0426] The resulting 268 and 504-base pair fragments are then combinedin a PCR using forward primer CH4(F) and reverse primer FAS (R), toproduce a fusion fragment of about 765 base pairs. This fragment isdigested with SacI and SalI, and the resulting 750-base pair fragment isgel purified. Plasmid pVBE also digested with SacI and SalI, and thelarger resulting fragment of about 6.8 Kb is gel purified. These twoSacII/SalI fragments are then ligated to produce CH4-Fas.

[0427] 16.3 Construction of CH4(TM)-Fas. An expression vector whichencodes a fusion protein comprising the human heavy chain CH1-CH4domains and the transmembrane domain of Cμ, fused to the death domain ofFas, designated herein as CH4(TM)-Fas, is constructed by the followingmethod. The fusion protein is illustrated in FIG. 13(c).

[0428] Plasmid pVHE, produced as described in Example 1. 1, is digestedwith BstEII and SalI and the smaller DNA fragment of about 1.4 Kb is gelpurified. This smaller fragment is then used as a template in a PCRreaction using forward primer CH4(F) as shown in section 16.2, andreverse primer CH4(R2) 5′ AATAGTGGTG ATATATTTCA CCTTGAACAA 3′ (SEQ IDNO:134). The resulting PCR product of about 356 base pairs is gelpurified.

[0429] A DNA fragment comprising the death domains of Fas is amplifiedfrom plasmid pBS-APO14.2 with forward primer FAS(F3)-5′TTGTTCAAGGTGAAAGTGAA GAGAAAGGAA 3′ (SEQ ID NO:135) and reverse primer FAS(R), asshown in section 16.1. The resulting PCR product of about 440 base pairsis gel purified.

[0430] The resulting 356 and 440-base pair fragments are then combinedin a PCR using forward primer CH4(F) and reverse primer FAS (R), toproduce a fusion fragment of about 795 base pairs. This fragment isdigested with SacII and SalI, and the resulting 780-base pair fragmentis gel purified. Plasmid pVHE also digested with SacII and SalI, and thelarger resulting fragment of about 6.8 Kb is gel purified. These twoSacII/SalI fragments are then ligated to produce CH4(TM)-Fas.

[0431] 16.4 Cloning and insertion of diverse VH genes into the Ig-Fasfusion proteins. Heavy chain variable region (VH) PCR products (aminoacids (−4) to (110)), produced as described in Example 1.4(a), using theprimers listed in Tables 1 and 2, are cloned into BssHII and BstEIIsites ov CH1-Fas, CH4-Fas and CH4(TM)-Fas. Because of the overlapbetween the CH1 domain sequence and the restriction enzyme sitesselected, this results in construction of a contiguous heavy chainfragment which lacks a functional signal peptide but remains in thecorrect translational reading frame.

Example 17 Generation of Igα and Igβ-Expressing HeLaS3 and COS7 CellLines

[0432] In order to express specific human monoclonal antibodies on thecell surface, heavy and light chain immunoglobulins must physicallyassociate with other proteins in the B cell receptor complex. Therefore,in order for host cells to be able to express the human antibody librarythey must be able to express the molecules and structures that arenecessary for the efficient synthesis and assembly of antibodies intomembrane-bound receptors. Mouse lymphoma cells express the molecules andstructures that are necessary for the expression of specific humanantibodies on the cell surface. However, one disadvantage of usinglymphoma cells for human antibody library expression is thatendogenously expressed immunoglobulin heavy and/or light chains canco-assemble with transgenic immunoglobulin chains, resulting in theformation of nonspecific heterogeneous molecules, which diluteantigen-specific receptors. Another disadvantage of using mouse lymphomacells to express the human antibody library is that vaccinia virusreplicates poorly in lymphocytic cell lines. Therefore, preferred celltypes for the expression of specific human antibodies are those whichpermit the generation of high titers of vaccinia virus and those thatare not derived from the B cell lineage. Preferred cell types includeHeLa cells, COS7 cells and BSC-1 cells.

[0433] The immunoglobulin heavy and light chains of the B cell receptorphysically associate with the heterodimer of the Igα and Igβtransmembrane proteins (Reth, M. 1992. Annu. Rev. Immunol. 10:97). Thisphysical association is necessary for the efficient transport ofmembrane-bound immunoglobulin to the cell surface and for thetransduction of signals through the B cell receptor (Venkitaraman, A. R.et al., 1991. Nature 352:777). However, it is unclear as to whetherIgα/Igβ heterodimers are necessary and sufficient for the expression ofmembrane-bound immunoglobulin in heterologous cell lines. Therefore, thecell surface expression of human antibodies on HeLaS3 and COS7 cells wasevaluated following their transfection with human Igα and Igβ cDNA.

[0434] 17.1 Cloning the human Igα and Igβ cDNA by PCR.

[0435] cDNA generated from the EBV-transformed human B cells was used asthe template in the PCR reactions to amplify human Igα and Igβ cDNA.Human Igα cDNA was amplified with the following primers:igα5′-5′ATTAGAATTCATGCCTGGGGGTCCAGGA3′, designated herein as (SEQ IDNO:136); and igα3′-5′ATTAGGATCCTCACGGCTTCTCCAGCTG3′, designated hereinas (SEQ ID NO:137).

[0436] Human Igβ cDNA was amplified with the following primers:igβ5′-5′ATTAGGATCCATGGCCAGGCTGGCGTTG3′, designated herein as (SEQ IDNO:138); and igβ3′-5′ATTACCAGCACACTGGTCACTCCTGGCCTGGGTG3′, designatedherein as (SEQ ID NO:139).

[0437] Products from Igα PCR reaction were cloned into pIRESneoexpression vector (Clontech) at EcoRI and BamHI sites, while those fromIgβ PCR reaction were cloned into pIREShyg vector (Clontech) at BamHIand BstXI sites. The identities of the cloned Igα and Igb were confirmedby DNA sequencing.

[0438] 17.2 Establishing Igα and Igβ-expressing HeLaS3 and COS7 stabletransfectants. HeLaS3 and COS7 cells (1×10⁶ per well in a 6-well plate)were transfected with 0.5 to 1 μg each of the purified pIRESneo-Igα andpIREShyg-Igβ plasmid DNA using the LIPOFECTAMINE PLUS Reagent (Lifetechnologies). Starting two days later, cells were selected with G418(at 0.4 mg/ml) and hygromycin B (at 0.2 mg/ml) for about 2 weeks.Drug-resistant HeLaS3 colonies were directly isolated and COS7transfectants were cloned by limiting dilution. The expression of Igoαand Igβ in each of these clones was then analyzed by RT-PCR, and theresults from the representative clones were as shown in FIG. 14.

Example 18 Construction of a Diverse Library of High Affinity HumanAntibodies

[0439] The current invention is the only available method for theconstruction of a diverse library of immunoglobulin genes in vaccinia orother pox viruses. The vaccinia vector can be designed to give highlevels of membrane receptor expression to allow efficient binding to anantigen coated matrix. Alternatively, the recombinant immunoglobulinheavy chain genes can be engineered to induce apoptosis uponcrosslinking of receptors by antigen. Since vaccinia virus can bereadily and efficiently recovered even from cells undergoing programmedcell death, the unique properties of this system make it possible torapidly select specific human antibody genes.

[0440] Optimal immunoglobulin heavy and light chains are selectedsequentially, which maximizes diversity by screening all available heavyand light chain combinations. The sequential screening strategy is to atfirst select an optimal heavy chain from a small library of 10⁵ H-chainrecombinants in the presence of a small library of 10⁴ diverse lightchains. This optimized H-chain is then used to select an optimizedpartner from a larger library of 10⁶ to 10⁷ recombinant L-chains. Oncean optimal L-chain is selected, it is possible to go back and select afurther optimized H-chain from a larger library of 10⁶ to 10⁷recombinant H-chains. This reiteration is a boot-strap strategy thatallows selection of a specific high-affinity antibody from as many as10¹⁴ H₂L₂ combinations. In contrast, selection of single chain Fv in aphage library or of Fab comprised of separate VH-CH1 and VL-CL genesencoded on a single plasmid is a one step process limited by thepractical size limit of a single phage library—perhaps 10¹¹ phageparticles.

[0441] Since it is not feasible to screen 10¹⁴ combinations of 10⁷ Hchains and 10⁷ L chains, the selection of optimal H chains begins from alibrary of 10⁵ H chain vaccinia recombinants in the presence of 10⁴ Lchains in a non-infectious vector. These combinations will mostly giverise to low affinity antibodies against a variety of epitopes and resultin selection of e.g., 1 to 100 different H chains. If 100 H chains areselected for a basic antibody, these can then be employed in a secondcycle of selection with a larger library of 10⁶ or 10⁷ vacciniarecombinant L chains to pick 100 optimal L chain partners. The originalH chains are then set aside and the 100 L chains are employed to selectnew, higher affinity H chains from a larger library of 10⁶ or 10⁷ Hchains.

[0442] The strategy is a kind of in vitro affinity maturation. As is thecase in normal immune responses, low affinity antibodies are initiallyselected and serve as the basis for selection of higher affinity progenyduring repeated cycles of immunization. Whereas higher affinity clonesmay be derived through somatic mutation in vivo, this in vitro strategyachieves the same end by the re-association of immunoglobulin chains. Inboth cases, the partner of the improved immunoglobulin chain is the sameas the partner in the original lower affinity antibody.

[0443] The basis of the strategy is leveraging the initial selection fora low affinity antibody. It is essential that a low affinity antibody beselected. The vaccinia-based method for sequential selection of H and Lchains is well-suited to insure that an initial low affinity selectionis successful because it has the avidity advantage that comes fromexpressing bivalent antibodies. In addition, the level of antibodyexpression can be regulated by employing different promoters in thevaccinia system. For example, the T7 polymerase system adapted tovaccinia gives high levels of expression relative to native vacciniapromoters. Initial rounds of selection can be based on a high level T7expression system to insure selection of a low affinity “basic antibody”and later rounds of selection can be based on low level expression todrive selection of a higher affinity derivative.

[0444] An outline of a method of the current invention for theconstruction of a diverse library of immunoglobulin genes in vaccinia isas follows:

[0445] 1. An immunoglobulin membrane associated heavy chain cDNA libraryis constructed from human lymphocytes in a vaccinia virus vectoraccording to the methods described herein. Specially engineered cells,for example CH33 cells, mouse myeloma cells, and human EBV transformedcell lines or, preferably, HeLa cells and other non-lymphoid cells thatdo not produce a competing immunoglobulin chain and efficiently supportvaccinia replication, are infected with the virus library at dilutionssuch than on average each cell is infected by one viral immunoglobulinheavy chain recombinant.

[0446] 2. These same cells are also infected with psoralin inactivatedimmunoglobulin light chain recombinant vaccinia virus from animmunoglobulin light chain library constructed in the same vacciniavirus vector. Alternatively, the cells may be transfected withimmunoglobulin light chain recombinants in a plasmid expression vector.In the population of cells as a whole, each heavy chain can beassociated with any light chain.

[0447] 3. The cells are incubated for a suitable period of time to allowoptimal expression of fully assembled antibodies on the cell surface.When the host cell is not of lymphoid origin, the efficiency of membraneantibody expression is enhanced by employing host cells for example,Hela or Cos 7 cells, that have been stably transfected with genes orcDNA expressing Igα and Igβ proteins.

[0448] 4a. The antigen of interest is bound to inert beads, which arethen mixed with the library of antibody expressing cells. Cells thatbind to antigen-coated beads are recovered and the associatedimmunoglobulin heavy chain recombinant virus is extracted.

[0449] 4b. Alternatively, a fluorescence tag is linked, directly orindirectly, to the antigen of interest. Antibody expressing cells whichbind the antigen are recovered by Fluorescence Activated Cell Sorting.

[0450] 4c. Alternatively, host cells may be employed in whichcross-linking of the antibody receptor with the antigen induces celldeath. This may occur naturally in host cells that are immature cells ofthe B cell lineage or it may be a consequence of incorporation of a Fasencoded death domain at the carboxyl terminus of the immunoglobulinheavy chain constant region. The lysed cells are separated from theliving cells and the recombinant viruses carrying the relevantimmunoglobulin heavy chains are extracted.

[0451] 5. The above cycle, steps 1-4, may be repeated multiple times,isolating recombinant virus each time and further enriching for heavychains that contribute to optimal antigen binding.

[0452] 6. Once specific antibody heavy chains have been selected, theentire procedure is repeated with an immunoglobulin light chain cDNAlibrary constructed in the proprietary vaccinia vector in order toselect the specific immunoglobulin light chains that contribute tooptimal antigen binding. Sequential selection of heavy and light chainsmaximizes diversity by screening all available heavy and light chaincombinations. The final MAb product is optimized by selection of a fullyassembled bivalent antibody rather than a single chain Fv or monomericFab.

[0453] 7. The MAb sequence is determined and specific binding verifiedthrough standard experimental techniques.

[0454] The final Mab product is optimized by selection of a fullyassembled bivalent antibody rather than a single chain Fv. That is,selection is based on bivalent (H₂L₂) antibodies rather than scFv or Fabfragments. Synthesis and assembly of fully human, complete antibodiesoccurs in mammalian cells allowing immunoglobulin chains to undergonormal post-translational modification and assembly. Synthesis andassembly of complete antibodies would likely be very inefficient inbacterial cells and many specificities are lost due to failure of manyantibodies to fold correctly in the abnormal physiological environmentof a bacterial cell.

[0455] A relatively wide range of antibody epitope specificities can beselected, including the selection of specificities on the basis offunctional activity. Specifically, antibodies can be selected on thebasis of specific physiological effects on target cells (e.g., screeningfor inhibition of TNF-secretion by activated monocytes; induction ofapoptosis; etc.) An outline of the method for screening for specific Mabon the basis of a functional assay is as follows:

[0456] 1. An immunoglobulin heavy chain cDNA library in secretory formis constructed from naive human lymphocytes in a vaccinia virus vectorprepared according to the methods described herein. Multiple pools of,for example, about 100 to about 1000 recombinant viruses, are separatelyexpanded and employed to infect producer cells at dilutions such that onaverage each cell is infected by one immunoglobulin heavy chainrecombinant virus. These same cells are also infected with psoralininactivated immunoglobulin light chain recombinant vaccinia virus froman immunoglobulin light chain library constructed in the same vacciniavirus vector. Alternatively, the infected cells may be transfected withimmunoglobulin light chain recombinants in a plasmid expression vector.In the population of cells as a whole, each heavy chain can beassociated with any light chain.

[0457] 2. Infected cells are incubated for a time sufficient to allowsecretion of fully assembled antibodies.

[0458] 3. Assay wells are set up in which indicator cells of functionalinterest are incubated in the presence of aliquots of secreted antibody.These might, for example, include activated monocytes secreting TNFα. Asimple ELISA assay for TNFα may then be employed to screen for any poolof antibodies that includes an activity that inhibits cytokinesecretion.

[0459] 4. Individual members of the selected pools are further analyzedto identify the relevant immunoglobulin heavy chain.

[0460] 5. Once specific antibody heavy chains have been selected, theentire procedure is repeated with an immunoglobulin light chain cDNAlibrary constructed in the proprietary vaccinia vector in order toselect specific immunoglobulin light chains that contribute to optimalantigen binding.

[0461] 6. The MAb sequences are identified and specific binding verifiedthrough standard experimental techniques. Because functional selectiondoes not require a priori knowledge of the target membrane receptor, theselected Mab is both a potential therapeutic and a discovery tool toidentify the relevant membrane receptor.

[0462] Selection occurs within human cell cultures following randomassociation of immunoglobulin heavy and light chains. As noted above,this avoids repertoire restrictions due to limitations of synthesis inbacteria. It also avoids restrictions of the antibody repertoire due totolerance to homologous gene products in mice. Mouse homologs ofimportant human proteins are often 80% to 85% identical to the humansequence. It should be expected, therefore, that the mouse antibodyresponse to a human protein would primarily focus on the 15% to 20% ofepitopes that are different in man and mouse. This invention allowsefficient selection of high affinity, fully human antibodies with abroad range of epitope specificities. The technology is applicable to awide variety of projects and targets including functional selection ofantibodies to previously unidentified membrane receptors with definedphysiological significance.

[0463] The present invention is not to be limited in scope by thespecific embodiments described which are intended as singleillustrations of individual aspects of the invention, and anyconstructs, viruses or enzymes which are functionally equivalent arewithin the scope of this invention. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

[0464] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thedisclosure and claims of U.S. application Ser. No. 08/935,377, filedSep. 22, 1997 and U.S. application Ser. No. 60/192,586, filed Mar. 28,2000 are herein incorporated by reference.

1 147 1 57 DNA Artificial Sequence p7.5/tk promoter 1 ggccaaaaattgaaaaacta gatctattta ttgcacgcgg ccgccatggg cccggcc 57 2 145 DNAArtificial Sequence p 7.5/ATG0/tk promoter 2 ggccaaaaat tgaaaaactagatctattta ttgcacgcgg ccgccgtgga tcccccgggc 60 tgcaggaatt cgatatcaagcttatcgata ccgtcgacct cgaggggggg cctaactaac 120 taattttgtt tttgtgggcccggcc 145 3 148 DNA Artificial Sequence p 7.5/ATG1/tk promoter 3ggccaaaaat tgaaaaacta gatctattta ttgcacgcgg ccgccatggt ggatcccccg 60ggctgcagga attcgatatc aagcttatcg ataccgtcga cctcgagggg gggcctaact 120aactaatttt gtttttgtgg gcccggcc 148 4 149 DNA Artificial Sequencep7.5/ATG2/tk vector 4 ggccaaaaat tgaaaaacta gatctattta ttgcacgcggccgccatgag tggatccccc 60 gggctgcagg aattcgatat caagcttatc gataccgtcgacctcgaggg ggggcctaac 120 taactaattt tgtttttgtg ggcccggcc 149 5 150 DNAArtificial Sequence p7.5/ATG3/tk vector 5 ggccaaaaat tgaaaaactagatctattta ttgcacgcgg ccgccatgac gtggatcccc 60 cgggctgcag gaattcgatatcaagcttat cgataccgtc gacctcgagg gggggcctaa 120 ctaactaatt ttgtttttgtgggcccggcc 150 6 15 PRT Artificial Sequence linker peptide 6 Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 7 15 PRTArtificial Sequence linker peptide 7 Glu Ser Gly Arg Ser Gly Gly Gly GlySer Gly Gly Gly Gly Ser 1 5 10 15 8 14 PRT Artificial Sequence linkerpeptide 8 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 109 15 PRT Artificial Sequence linker peptide 9 Glu Gly Lys Ser Ser GlySer Gly Ser Glu Ser Lys Ser Thr Gln 1 5 10 15 10 14 PRT ArtificialSequence linker peptide 10 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu SerLys Val Asp 1 5 10 11 14 PRT Artificial Sequence linker peptide 11 GlySer Thr Ser Gly Ser Gly Lys Ser Ser Glu Gly Lys Gly 1 5 10 12 18 PRTArtificial Sequence linker peptide 12 Lys Glu Ser Gly Ser Val Ser SerGlu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp 13 16 PRT ArtificialSequence linker peptide 13 Glu Ser Gly Ser Val Ser Ser Glu Glu Leu AlaPhe Arg Ser Leu Asp 1 5 10 15 14 1555 DNA Artificial Sequence pVHEtransfer plasmid 14 ggccaaaaat tgaaaaacta gatctattta ttgcacgcggccgcaaacca tgggatggag 60 ctgtatcatc ctcttcttgg tagcaacagc tacaggcgcgcatatggtca ccgtctcctc 120 agggagtgca tccgccccaa cccttttccc cctcgtctcctgtgagaatt ccccgtcgga 180 tacgagcagc gtggccgttg gctgcctcgc acaggacttccttcccgact ccatcacttt 240 ctcctggaaa tacaagaaca actctgacat cagcagcacccggggcttcc catcagtcct 300 gagagggggc aagtacgcag ccacctcaca ggtgctgctgccttccaagg acgtcatgca 360 gggcacagac gaacacgtgg tgtgcaaagt ccagcaccccaacggcaaca aagaaaagaa 420 cgtgcctctt ccagtgattg ctgagctgcc tcccaaagtgagcgtcttcg tcccaccccg 480 cgacggcttc ttcggcaacc cccgcagcaa gtccaagctcatctgccagg ccacgggttt 540 cagtccccgg cagattcagg tgtcctggct gcgcgaggggaagcaggtgg ggtctggcgt 600 caccacggac caggtgcagg ctgaggccaa agagtctgggcccacgacct acaaggtgac 660 tagcacactg accatcaaag agagcgactg gctcagccagagcatgttca cctgccgcgt 720 ggatcacagg ggcctgacct tccagcagaa tgcgtcctccatgtgtgtcc ccgatcaaga 780 cacagccatc cgggtcttcg ccatcccccc atcctttgccagcatcttcc tcaccaagtc 840 caccaagttg acctgcctgg tcacagacct gaccacctatgacagcgtga ccatctcctg 900 gacccgccag aatggcgaag ctgtgaaaac ccacaccaacatctccgaga gccaccccaa 960 tgccactttc agcgccgtgg gtgaggccag catctgcgaggatgactgga attccgggga 1020 gaggttcacg tgcaccgtga cccacacaga cctgccctcgccactgaagc agaccatctc 1080 ccggcccaag ggggtggccc tgcacaggcc cgatgtctacttgctgccac cagcccggga 1140 gcagctgaac ctgcgggagt cggccaccat cacgtgcctggtgacgggct tctctcccgc 1200 ggacgtcttc gtgcagtgga tgcagagggg gcagcccttgtccccggaga agtatgtgac 1260 cagcgcccca atgcctgagc cccaggcccc aggccggtacttcgcccaca gcatcctgac 1320 cgtgtccgaa gaggaatgga acacggggga gacctacacctgcgtggtgg cccatgaggc 1380 cctgcccaac agggtcactg agaggaccgt ggacaagtccaccgaggggg aggtgagcgc 1440 cgacgaggag ggctttgaga acctgtgggc caccgcctccaccttcatcg tcctcttcct 1500 cctgagcctc ttctacagta ccaccgtcac cttgttcaaggtgaaatgag tcgac 1555 15 6 DNA Artificial Sequence unique BssHII site inpVHE 15 gcgcgc 6 16 7 DNA Artificial Sequence Unique BstEII site in pVHE16 ggtcacc 7 17 446 DNA Artificial Sequence pVKE transfer plasmid 17ggccaaaaat tgaaaaacta gatctattta ttgcacgcgg ccgcccatgg gatggagctg 60tatcatcctc ttcttggtag caacagctac aggcgtgcac ttgactcgag atcaaacgaa 120ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa 180ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga 240aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca 300aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac 360acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct 420tcaacagggg agagtgttag gtcgac 446 18 6 DNA Artificial Sequence uniqueApaLI site in pVKE plasmid 18 gtgcac 6 19 6 DNA Artificial Sequenceunique XhoI site in pVKE plasmid 19 ctcgag 6 20 455 DNA ArtificialSequence pVLE transfer plasmid 20 ggccaaaaat tgaaaaacta gatctatttattgcacgcgg ccgcccatgg gatggagctg 60 tatcatcctc ttcttggtag caacagctacaggcgtgcac ttgactcgag aagcttaccg 120 tcctacgaac tgtggctgca ccatctgtcttcatcttccc gccatctgat gagcagttga 180 aatctggaac tgcctctgtt gtgtgcctgctgaataactt ctatcccaga gaggccaaag 240 tacagtggaa ggtggataac gccctccaatcgggtaactc ccaggagagt gtcacagagc 300 aggacagcaa ggacagcacc tacagcctcagcagcaccct gacgctgagc aaagcagact 360 acgagaaaca caaagtctac gcctgcgaagtcacccatca gggcctgagc tcgcccgtca 420 caaagagctt caacagggga gagtgttaggtcgac 455 21 6 DNA Artificial Sequence Unique ApaLI site in pVLE plasmid21 gtgcac 6 22 6 DNA Artificial Sequence Unique HindIII site in pVLE 22aagctt 6 23 9 PRT Artificial Sequence H-2Kd restricted peptide 23 GlyTyr Lys Ala Gly Met Ile His Ile 1 5 24 29 DNA Artificial Sequence primer24 attaggatcc ggtcaccgtc tcctcaggg 29 25 34 DNA Artificial Sequenceprimer 25 attagtcgac tcatttcacc ttgaacaagg tgac 34 26 47 DNA ArtificialSequence cassette used to generate p7.5/tk2 26 gcggccgcaa accatggaaagcgcgcatat ggtcaccaaa agtcgac 47 27 29 DNA Artificial Sequence primer 27attaggatcc ggtcaccgtc tcctcaggg 29 28 31 DNA Artificial Sequence primer28 attagtcgac tcagtagcag gtgccagctg t 31 29 50 DNA Artificial Sequencecassette used to generate p7.5/tk3 29 gcggccgccc atggatacgt gcacttgactcgagaagctt agtagtcgac 50 30 30 DNA Artificial Sequence primer 30caggactcga gatcaaacga actgtggctg 30 31 39 DNA Artificial Sequence primer31 aatatgtcga cctaacactc tcccctgttg aagctcttt 39 32 38 DNA ArtificialSequence primer 32 aatatgtcga cctaacactc tcccctgttg aagctctt 38 33 40DNA Artificial Sequence primer 33 atttaagctt accgtcctac gaactgtggctgcaccatct 40 34 38 DNA Artificial Sequence primer 34 ttttgcgcgcactcccaggt gcagctggtg cagtctgg 38 35 38 DNA Artificial Sequence primer35 ttttgcgcgc actccgaggt gcagctggtg gagtctgg 38 36 38 DNA ArtificialSequence primer 36 ttttgcgcgc actcccaggt gcagctgcag gagtcggg 38 37 27DNA Artificial Sequence primer 37 gacggtgacc agggtgccct ggcccca 27 38 27DNA Artificial Sequence primer 38 gacggtgacc agggtgccac ggcccca 27 39 27DNA Artificial Sequence primer 39 gacggtgacc attgtccctt ggcccca 27 40 27DNA Artificial Sequence primer 40 gacggtgacc agggttccct ggcccca 27 41 27DNA Artificial Sequence primer 41 gacggtgacc gtggtccctt ggcccca 27 42 35DNA Artificial Sequence primer 42 tttgtgcact ccgacatcca gatgacccag tctcc35 43 35 DNA Artificial Sequence primer 43 tttgtgcact ccgatgttgtgatgactcag tctcc 35 44 35 DNA Artificial Sequence primer 44 tttgtgcactccgaaattgt gttgacgcag tctcc 35 45 35 DNA Artificial Sequence primer 45tttgtgcact ccgacatcgt gatgacccag tctcc 35 46 35 DNA Artificial Sequenceprimer 46 tttgtgcact ccgaaacgac actcacgcag tctcc 35 47 35 DNA ArtificialSequence primer 47 tttgtgcact ccgaaattgt gctgactcag tctcc 35 48 27 DNAArtificial Sequence primer 48 gatctcgagc ttggtccctt ggccgaa 27 49 27 DNAArtificial Sequence primer 49 gatctcgagc ttggtcccct ggccaaa 27 50 27 DNAArtificial Sequence primer 50 gatctcgagt ttggtcccag ggccgaa 27 51 27 DNAArtificial Sequence primer 51 gatctcgagc ttggtccctc cgccgaa 27 52 27 DNAArtificial Sequence primer 52 aatctcgagt cgtgtccctt ggccgaa 27 53 35 DNAArtificial Sequence primer 53 tttgtgcact cccagtctgt gttgacgcag ccgcc 3554 35 DNA Artificial Sequence primer 54 tttgtgcact cccagtctgc cctgactcagcctgc 35 55 35 DNA Artificial Sequence primer 55 tttgtgcact cctcctatgtgctgactcag ccacc 35 56 35 DNA Artificial Sequence primer 56 tttgtgcactcctcttctga gctgactcag gaccc 35 57 35 DNA Artificial Sequence primer 57tttgtgcact cccacgttat actgactcaa ccgcc 35 58 35 DNA Artificial Sequenceprimer 58 tttgtgcact cccaggctgt gctcactcag ccgtc 35 59 35 DNA ArtificialSequence primer 59 tttgtgcact ccaattttat gctgactcag cccca 35 60 35 DNAArtificial Sequence primer 60 tttgtgcact cccaggctgt ggtgactcag gagcc 3561 27 DNA Artificial Sequence primer 61 ggtaagcttg gtcccagttc cgaagac 2762 25 DNA Artificial Sequence primer 62 ggtaagcttg gtccctccgc cgaat 2563 39 DNA Artificial Sequence primer 63 aatatgcgcg cactcccagg tgcagctggtgcagtctgg 39 64 39 DNA Artificial Sequence primer 64 aatatgcgcgcactcccagg tcaccttgaa ggagtctgg 39 65 39 DNA Artificial Sequence primer65 aatatgcgcg cactccgagg tgcagctggt ggagtctgg 39 66 39 DNA ArtificialSequence primer 66 aatatgcgcg cactcccagg tgcagctgca ggagtcggg 39 67 38DNA Artificial Sequence primer 67 aatatgcgcg cactccgagg tgcagctggtgcagtctg 38 68 29 DNA Artificial Sequence primer 68 gagacggtgaccagggtgcc ctggcccca 29 69 29 DNA Artificial Sequence primer 69gagacggtga ccagggtgcc acggcccca 29 70 29 DNA Artificial Sequence primer70 gagacggtga ccattgtccc ttggcccca 29 71 29 DNA Artificial Sequenceprimer 71 gagacggtga ccagggttcc ctggcccca 29 72 29 DNA ArtificialSequence primer 72 gagacggtga ccgtggtccc ttggcccca 29 73 37 DNAArtificial Sequence primer 73 caggagtgca ctccgacatc cagatgaccc agtctcc37 74 37 DNA Artificial Sequence primer 74 caggagtgca ctccgatgttgtgatgactc agtctcc 37 75 37 DNA Artificial Sequence primer 75 caggagtgcactccgaaatt gtgttgacgc agtctcc 37 76 37 DNA Artificial Sequence primer 76caggagtgca ctccgacatc gtgatgaccc agtctcc 37 77 37 DNA ArtificialSequence primer 77 caggagtgca ctccgaaacg acactcacgc agtctcc 37 78 37 DNAArtificial Sequence primer 78 caggagtgca ctccgaaatt gtgctgactc agtctcc37 79 29 DNA Artificial Sequence primer 79 ttgatctcga gcttggtcccttggccgaa 29 80 29 DNA Artificial Sequence primer 80 ttgatctcgagcttggtccc ctggccaaa 29 81 29 DNA Artificial Sequence primer 81ttgatctcga gtttggtccc agggccgaa 29 82 29 DNA Artificial Sequence primer82 ttgatctcga gcttggtccc tccgccgaa 29 83 29 DNA Artificial Sequenceprimer 83 ttaatctcga gtcgtgtccc ttggccgaa 29 84 37 DNA ArtificialSequence primer 84 cagatgtgca ctcccagtct gtgttgacgc agccgcc 37 85 37 DNAArtificial Sequence primer 85 cagatgtgca ctcccagtct gccctgactc agcctgc37 86 37 DNA Artificial Sequence primer 86 cagatgtgca ctcctcctatgtgctgactc agccacc 37 87 37 DNA Artificial Sequence primer 87 cagatgtgcactcctcttct gagctgactc aggaccc 37 88 37 DNA Artificial Sequence primer 88cagatgtgca ctcccacgtt atactgactc aaccgcc 37 89 37 DNA ArtificialSequence primer 89 cagatgtgca ctcccaggct gtgctcactc agccgtc 37 90 37 DNAArtificial Sequence primer 90 cagatgtgca ctccaatttt atgctgactc agcccca37 91 37 DNA Artificial Sequence primer 91 cagatgtgca ctcccaggctgtggtgactc aggagcc 37 92 29 DNA Artificial Sequence primer 92 acggtaagcttggtcccagt tccgaagac 29 93 29 DNA Artificial Sequence primer 93acggtaagct tggtccctcc gccgaatac 29 94 21 DNA Artificial Sequence primer94 atgttacgtc ctgtagaaac c 21 95 21 DNA Artificial Sequence primer 95tcattgtttg cctccctgct g 21 96 28 DNA Artificial Sequence primer 96aaagcggccg ccccgggatg ttacgtcc 28 97 29 DNA Artificial Sequence primer97 aaagggcccg gcgcgcctca ttgtttgcc 29 98 37 DNA Artificial Sequenceprimer 98 aaaggatcca taatgaattc agtgactgta tcacacg 37 99 34 DNAArtificial Sequence primer 99 cttgcggccg cttaataaat aaacccttga gccc 34100 34 DNA Artificial Sequence primer 100 attgagctct taatacttttgtcgggtaac agag 34 101 29 DNA Artificial Sequence primer 101 ttactcgagagtgtcgcaat ttggatttt 29 102 29 DNA Artificial Sequence primer 102aaagaattcc tttattgtca tcggccaaa 29 103 30 DNA Artificial Sequence primer103 aatctgcagt cattgtttgc ctccctgctg 30 104 37 DNA Artificial Sequenceprimer 104 aaagaattca taatgaattc agtgactgta tcacacg 37 105 32 DNAArtificial Sequence primer 105 cttggatcct taataaataa acccttgagc cc 32106 27 DNA Artificial Sequence primer 106 aataagcttt actccagata atatgga27 107 23 DNA Artificial Sequence primer 107 aatctgcagc ccagttccat ttt23 108 23 DNA Artificial Sequence primer 108 aatggatcct catccagcgg cta23 109 27 DNA Artificial Sequence primer 109 aatgagctct agtacctacaacccgaa 27 110 28 DNA Artificial Sequence primer 110 aaagtcgacggccaaaaatt gaaatttt 28 111 25 DNA Artificial Sequence primer 111aatggatcct cattgtttgc ctccc 25 112 51 DNA Artificial Sequence cassetteconverting Plasmid p7.5/tk3 to p7.5/tk3.1 112 gcggccgccc atggatagcgtgcacttgac tcgagaagct tagtagtcga c 51 113 22 DNA Artificial Sequenceregion substituted to convert plasmid p7.5/tk3.1 top7.5/tk3.2 113ctcgagaagc ttagtagtcg ac 22 114 78 DNA Artificial Sequence cassette forthe conversion of plasmid p7.5/tk3.1 to p7.5/tk3.2 114 ctcgagatcaaagagggtaa atcttccgga tctggttccg aaggcgcgca tgcggtcacc 60 gtctcctcatgagtcgac 78 115 42 DNA Artificial Sequence p7.5/tk3.2 linker 115gagggtaaat cttccggatc tggttccgaa ggcgcgcact cc 42 116 14 PRT ArtificialSequence p7.5/tk3.2 linker 116 Glu Gly Lys Ser Ser Gly Ser Gly Ser GluGly Ala His Ser 1 5 10 117 16 DNA Artificial Sequence region substitutedto convert plasmid p7.5/tk3.1 to p7.5/tk3.3 117 aagcttagta gtcgac 16 11881 DNA Artificial Sequence cassette for the conversion of plasmidp7.5/tk3.1 to p7.5/tk3.3 118 aagcttaccg tcctagaggg taaatcttcc ggatctggttccgaaggcgc gcatgcggtc 60 accgtctcct catgagtcga c 81 119 42 DNAArtificial Sequence p7.5/tk3.3 linker 119 gagggtaaat cttccggatctggttccgaa ggcgcgcact cc 42 120 14 PRT Artificial Sequence p7.5/tk3.3linker 120 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Gly Ala His Ser 1 510 121 29 DNA Artificial Sequence primer 121 attaggatcc ggtcaccgtctcctcagcc 29 122 34 DNA Artificial Sequence primer 122 attagtcgactcatttaccc ggagacaggg agag 34 123 24 DNA Artificial Sequence primer 123aatatggtca ccgtctcctc agcc 24 124 36 DNA Artificial Sequence primer 124mnnmnnmnnm nnmnnmnntt caggtgctgg gcacgg 36 125 36 DNA ArtificialSequence primer 125 nnknnknnkn nknnknnkgt cttcctcttc ccccca 36 126 23DNA Artificial Sequence primer 126 aatatgtcga ctcatttacc cgg 23 127 28DNA Artificial Sequence primer 127 acacggtcac cgtctcctca gggagtgc 28 12831 DNA Artificial Sequence primer 128 agttagatct ggatcctgga agaggcacgt t31 129 30 DNA Artificial Sequence primer 129 aacgtgcctc ttccaggatccagatctaac 30 130 33 DNA Artificial Sequence primer 130 acgcgtcgacctagaccaag ctttggattt cat 33 131 21 DNA Artificial Sequence primer 131ctctcccgcg gacgtcttcg t 21 132 31 DNA Artificial Sequence primer 132agttagatct ggatccctca aagccctcct c 31 133 30 DNA Artificial Sequenceprimer 133 gaggagggct ttgagggatc cagatctaac 30 134 30 DNA ArtificialSequence primer 134 aatagtggtg atatatttca ccttgaacaa 30 135 30 DNAArtificial Sequence primer 135 ttgttcaagg tgaaagtgaa gagaaaggaa 30 13628 DNA Artificial Sequence primer 136 attagaattc atgcctgggg gtccagga 28137 28 DNA Artificial Sequence primer 137 attaggatcc tcacggcttc tccagctg28 138 28 DNA Artificial Sequence primer 138 attaggatcc atggccaggctggcgttg 28 139 34 DNA Artificial Sequence primer 139 attaccagcacactggtcac tcctggcctg ggtg 34 140 69 DNA Artificial Sequence p7.5/tkpromoter 140 ggccaaaaat tgaaaaacta gatctattta ttgcacgcgg ccgcc atg ggcccg gcc 57 Met Gly Pro Ala 1 gcc aac ggc gga 69 Ala Asn Gly Gly 5 141 8PRT Artificial Sequence tk sequence of p7.5/tk 141 Met Gly Pro Ala AlaAsn Gly Gly 1 5 142 75 DNA Artificial Sequence pE/Ltk promoter 142ggccaaaaat tgaaatttta tttttttttt ttggaatata aagcggccgc c atg ggc 57 MetGly 1 ccg gcc gcc aac ggc gga 75 Pro Ala Ala Asn Gly Gly 5 143 8 PRTArtificial Sequence tk sequence of pE/Ltk 143 Met Gly Pro Ala Ala AsnGly Gly 1 5 144 39 DNA Artificial Sequence Primer 144 aatatgcgcgcactcccagg tcaccttgaa ggagtctgg 39 145 38 DNA Artificial Sequence Primer145 aatatgcgcg cactccgagg tgcagctggt gcagtctg 38 146 19 PRT ArtificialSequence Signal Sequence 146 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu ValAla Thr Ala Thr Gly 1 5 10 15 Ala His Ser 147 26 PRT Artificial SequenceSignal Sequence 147 Asn Leu Trp Thr Thr Ala Ser Thr Phe Ile Val Leu PheLeu Leu Ser 1 5 10 15 Leu Phe Tyr Ser Thr Thr Val Thr Leu Phe 20 25

What is claimed is:
 1. A method of selecting polynucleotides whichencode an antigen-specific immunoglobulin molecule, or antigen-specificfragment thereof, comprising: (a) introducing into a population ofeukaryotic host cells capable of expressing said immunoglobulin moleculea first library of polynucleotides encoding, through operableassociation with a transcriptional control region, aplurality of firstimmunoglobulin subunit polypeptides, each first immunoglobulin subunitpolypeptide comprising: (i) a first immunoglobulin constant regionselected from the group consisting of a heavy chain constant region anda light chain constant region, (ii) an imrnmunoglobulin variable regioncorresponding to said first constant region, and (iii) a signal peptidecapable of directing cell surface expression or secretion of said firstimmunoglobulin subunit polypeptide; (b) introducing into said host cellsa second library of polynucleotides encoding, through operableassociation with a transcriptional control region, a plurality of secondimmunoglobulin subunit polypeptides, each comprising: (i) a secondimmunoglobulin constant region selected from the group consisting of aheavy chain constant region and a light chain constant region, whereinsaid second immunoglobulin constant region is not the same as said firstimmunoglobulin constant region, (ii) an immunoglobulin variable regioncorresponding to said second constant region, and (iii) a signal peptidecapable of directing cell surface expression or secretion of said secondimmunoglobulin subunit polypeptide, wherein said second immunoglobulinsubunit polypeptide is capable of combining with said firstimmunoglobulin subunit polypeptide to form an immunoglobulin molecule,or antigen-specific fragment thereof, attached to the membrane surfaceof said host cells; (c) permitting expression of immunoglobulinmolecules, or antigen-specific fragments thereof, from said host cells;(d) contacting said immunoglobulin molecules with an antigen; and (e)recovering those polynucleotides of said first library which expressimmunoglobulin molecules, or antigen-specific fragments thereof,specific for said antigen.
 2. The method of claim 1, further comprising:(f) introducing said recovered polynucleotides into a population of hostcells capable of expressing said immunoglobulin molecule; (g)introducing into said host cells said second library of polynucleotides;(h) permitting expression of immunoglobulin molecules, orantigen-specific fragments thereof, from said host cells; (i) contactingsaid host cells with said antigen; and (j) recovering thosepolynucleotides of said first library which express immunoglobulinmolecules, or antigen-specific fragments thereof, specific for saidantigen.
 3. The method of claim 2, further comprising repeating steps(f)-(j) one or more times, thereby enriching for polynucleotides of saidfirst library which encode a first immunoglobulin subunit polypeptidewhich, as part of an immunoglobulin molecule, or antigen-specificfragment thereof, specifically binds said antigen.
 4. The method ofclaim 1, further comprising isolating those polynucleotides recoveredfrom said first library.
 5. The method of claim 4, further comprising:(k) introducing into a population of eukaryotic host cells capable ofexpressing said immunoglobulin molecule said second library ofpolynucleotides; (l) introducing into said host cells thosepolynucleotides isolated from said first library; (m) permittingexpression of immunoglobulin molecules, or antigen-specific fragmentsthereof, from said host cells; (n) contacting said host cells with saidspecific antigen; and (o) recovering those polynucleotides of saidsecond library which express immunoglobulin molecules, orantigen-specific fragments thereof, specific for said antigen.
 6. Themethod of claim 5, further comprising: (p) introducing said recoveredpolynucleotides into a population of host cells capable of expressingsaid immunoglobulin molecule; (q) introducing into said host cells thosepolynucleotides isolated from said first library; (r) permittingexpression of immunoglobulin molecules, or antigen-specific fragmentsthereof, from said host cells; (s) contacting said host cells with saidantigen; and (t) recovering those polynucleotides of said second librarywhich express immunoglobulin molecules, or antigen-specific fragmentsthereof, specific for said antigen.
 7. The method of claim 6, furthercomprising repeating steps (p)-(t) one or more times, thereby enrichingfor polynucleotides of said second library which encode a secondimmunoglobulin subunit polypeptide which, as part of an immunoglobulinmolecule, or antigen-specific fragment thereof, specifically binds saidantigen.
 8. The method of claim 7, further comprising isolating thosepolynucleotides recovered from said second library.
 9. The method ofclaim 1, wherein said immunoglobulin molecule is a human immunoglobulinmolecule.
 10. The method of claim 1, wherein said first immunoglobulinsubunit polypeptide is an immunoglobulin heavy chain, orantigen-specific fragment thereof.
 11. The method of claim 10, whereinsaid immunoglobulin heavy chain, or antigen-specific fragment thereof,is a membrane bound form of an immunoglobulin heavy chain.
 12. Themethod of claim 11, wherein said immunoglobulin heavy chain, orantigen-specific fragment thereof, comprises a naturally-occurringimmunoglobulin transmembrane domain.
 13. The method of claim 11, whereinsaid immunoglobulin heavy chain, or antigen-specific fragment thereof,is attached to said host cell as part of a fusion protein.
 14. Themethod of claim 13, wherein said fusion protein comprises a heterologoustransmembrane domain.
 15. The method of claim 13, wherein said fusionprotein comprises a fas death domain.
 16. The method of claim 10,wherein said immunoglobulin heavy chain, or antigen-specific fragmentthereof, is selected from the group consisting of an IgM heavy chain, anIgD heavy chain, an IgG heavy chain, an IgA heavy chain, an IgE heavychain, and an antigen-specific fragment of any of said heavy chains. 17.The method of claim 10, wherein said immunoglobulin heavy chain constantregion sequence comprises a modification that supports an altered immuneeffector function.
 18. The method of claim 16, wherein saidimmunoglobulin heavy chain, or antigen-specific fragment thereof,comprises an IgM heavy chain, or an antigen specific fragment thereof.19. The method of claim 1, wherein said second immunoglobulin subunitpolypeptide is an immunoglobulin light chain, or antigen-specificfragment thereof.
 20. The method of claim 19, wherein saidimmunoglobulin light chain, or antigen-specific fragment thereof,associates with said immunoglobulin heavy chain, or antigen-specificfragment thereof, thereby producing a immunoglobulin molecule, orantigen-specific fragment thereof.
 21. The method of claim 19, whereinsaid immunoglobulin light chain is selected from the group consisting ofa kappa light chain and a lambda light chain.
 22. The method of claim 1,wherein said first library of polynucleotides is constructed in aeukaryotic virus vector.
 23. The method of claim 1, wherein said secondlibrary of polynucleotides is constructed in a eukaryotic virus vector.24. The method of claim 5, wherein said polynucleotides isolated fromsaid first library are introduced by means of a eukaryotic virus vector.25. The method of claim 1, wherein said second library ofpolynucleotides is constructed in a plasmid vector.
 26. The method ofclaim 22, wherein said host cells are infected with said first libraryat an MOI ranging from about 1 to about 10, and wherein said secondlibrary is introduced under conditions which allow up to 20polynucleotides of said second library to be taken up by each infectedhost cell.
 27. The method of claim 5, wherein said polynucleotidesisolated from said first library are introduced into said host cells ina plasmid vector.
 28. The method of claim 22, wherein said eukaryoticvirus vector is an animal virus vector.
 29. The method of claim 23,wherein said eukaryotic virus vector is an animal virus vector.
 30. Themethod of claim 28, wherein said virus vector is capable of producinginfectious viral particles in mammalian cells.
 31. The method of claim30, wherein the naturally-occurring genome of said virus vector is DNA.32. The method of claim 30, wherein the naturally-occurring genome ofsaid virus vector is RNA.
 33. The method of claim 31, wherein thenaturally-occurring genome of said virus vector is linear,double-stranded DNA.
 34. The method of claim 33, wherein said virusvector is selected from the group consisting of an adenovirus vector, aherpesvirus vector and a poxvirus vector.
 35. The method of claim 34,wherein said virus vector is a poxvirus vector.
 36. The method of claim35, wherein said poxvirus vector is selected from the group consistingof an orthopoxvirus vector, an avipoxvirus vector, a capripoxvirusvector, a leporipoxvirus vector, an entomopoxvirus vector, and asuipoxvirus vector.
 37. The method of claim 36, wherein said poxvirusvector is an orthopoxvirus vector selected from the group consisting ofa vaccinia virus vector and a raccoon poxvirus vector.
 38. The method ofclaim 37, wherein said animal virus vector is a vaccinia virus vector.39. The method of claim 38, wherein said host cells are permissive forthe production of infectious viral particles of said virus.
 40. Themethod of claim 38, wherein said vaccinia virus is attenuated.
 41. Themethod of claim 40, wherein said vaccinia virus vector is deficient inD4R synthesis.
 42. The method of claim 35, wherein said transcriptionalcontrol region of said first library of polynucleotides functions in thecytoplasm of a poxvirus-infected cell.
 43. The method of claim 25,wherein said plasmid vector directs synthesis of said secondimmunoglobulin subunit in the cytoplasm of a poxvirus-infected cellthrough operable association with a transcription control region. 44.The method of claim 42, wherein said transcriptional control regioncomprises a promoter.
 45. The method of claim 44, wherein said promoteris constitutive.
 46. The method of claim 45, wherein said promoter is avaccinia virus p7.5 promoter.
 47. The method of claim 45, wherein saidpromoter is a synthetic early/late promoter.
 48. The method of claim 44,wherein said promoter is a T7 phage promoter active in cells in which T7RNA polymerase is expressed.
 49. The method of claim 42, wherein saidtranscriptional control region comprises a transcriptional terminationregion.
 50. The method of claim 22, wherein said first library ofpolynucleotides is constructed in a eukaryotic virus vector by a methodcomprising: (a) cleaving an isolated linear DNA virus genome to producea first viral fragment and a second viral fragment, wherein said firstfragment is nonhomologous with said second fragment; (b) providing apopulation of transfer plasmids comprising said polynucleotides whichencode said plurality of immunoglobulin heavy chains through operableassociation with a transcription control region, flanked by a 5′flanking region and a 3′ flanking region, wherein said 5′ flankingregion is homologous to said first viral fragment and said 3′ flankingregion is homologous to said second viral fragment; and wherein saidtransfer plasmids are capable of homologous recombination with saidfirst and second viral fragments such that a viable virus genome isformed; (c) introducing said transfer plasmids and said first and secondviral fragments into a host cell under conditions wherein a transferplasmid and said viral fragments undergo in vivo homologousrecombination, thereby producing a viable modified virus genomecomprising a polynucleotide which encodes an immunoglobulin heavy chain;and (d) recovering said modified virus genome.
 51. The method of claim23, wherein said second library of polynucleotides is constructed in aeukaryotic virus vector by a method comprising: (a) cleaving an isolatedlinear DNA virus genome to produce a first viral fragment and a secondviral fragment, wherein said first fragment is nonhomologous with saidsecond fragment; (b) providing a population of transfer plasmidscomprising said polynucleotides which encode said plurality ofimmunoglobulin light chains through operable association with atranscription control region, flanked by a 5′ flanking region and a3′flanking region, wherein said 5′ flanking region is homologous to saidfirst viral fragment and said 3′ flanking region is homologous to saidsecond viral fragment; and wherein said transfer plasmids are capable ofhomologous recombination with said first and second viral fragments suchthat a viable virus genome is formed; (c) introducing said transferplasmids and said first and second viral fragments into a host cellunder conditions wherein a transfer plasmid and said viral fragmentsundergo in vivo homologous recombination, thereby producing a viablemodified virus genome comprising a polynucleotide which encodes animmunoglobulin light chain; and (d) recovering said modified virusgenome.
 52. The method of claim 1, wherein said polynucleotides encodingantigen-specific immunoglobulin molecules are identified throughdetection of an effect selected from the group consisting of: (a)antigen-induced cell death; (b) antigen-induced signaling; and (c)antigen-specific binding.
 53. The method of claim 5, wherein saidpolynucleotides encoding antigen-specific immunoglobulin molecules areidentified through detection of an effect selected from the groupconsisting of: (a) antigen-induced cell death; (b) antigen-inducedsignaling; and (c) antigen-specific binding.
 54. The method of claim 52,wherein said effect is antigen-induced cell death.
 55. The method ofclaim 53, wherein said effect is antigen-induced cell death.
 56. Themethod of claim 54, wherein said host cells express immunoglobulinmolecules on their surface, and wherein said host cells expressingimmunoglobulin molecules which bind said antigen directly respond tocross-linking of antigen-specific immunoglobulin receptors by inductionof apoptosis.
 57. The method of claim 55, wherein said host cellsexpress immunoglobulin molecules on their surface, and wherein said hostcells expressing immunoglobulin molecules which bind said antigendirectly respond to cross-linking of antigen-specific immunoglobulinreceptors by induction of apoptosis.
 58. The method of claim 52, whereinsaid effect is antigen-induced signaling.
 59. The method of claim 53,wherein said effect is antigen-induced signaling.
 60. The method ofclaim 58, wherein said host cells express immunoglobulin molecules ontheir surface, and wherein said host cells expressing immunoglobulinmolecules which bind said antigen respond to cross-linking ofantigen-specific immunoglobulin receptors by expression of a detectablereporter molecule.
 61. The method of claim 59, wherein said host cellsexpress immunoglobulin molecules on their surface, and wherein said hostcells expressing immunoglobulin molecules which bind said antigenrespond to cross-linking of antigen-specific immunoglobulin receptors byexpression of a detectable reporter molecule.
 62. The method of claim60, wherein said reporter molecule is selected from the group consistingof luciferase, green fluorescent protein, and beta-galactosidase. 63.The method of claim 61, wherein said reporter molecule is selected fromthe group consisting of luciferase, green fluorescent protein, andbeta-galactosidase.
 64. The method of claim 52, wherein said effect isantigen-specific binding.
 65. The method of claim 64, comprising: (a)contacting pools of said host cells with said antigen under conditionswherein antigen-specific immunoglobulin molecules expressed by said hostcells will bind to said antigen; and (b) recovering polynucleotides ofsaid first library from those host cell pools, or from replicate poolsof polynucleotides set aside previously, expressing immunoglobulinmolecules to which said antigen was bound.
 66. The method of claim 65,further comprising: (c) dividing said recovered polynucleotides into aplurality of sub-pools and introducing said sub-pools into populationsof host cells capable of expressing said immunoglobulin molecule; (d)permitting expression of immunoglobulin molecules, or antigen-specificfragments thereof, from said host cells; (e) contacting said pools withsaid antigen under conditions wherein antigen-specific immunoglobulinmolecules expressed by said host cells bind to said antigen; and (f)recovering polynucleotides of said first library from those host cellpools, or from replicate pools of polynucleotides set aside previously,expressing immunoglobulin molecules to which said antigen was bound. 67.The method of claim 66, further comprising repeating steps (c)-(f) oneor more times, thereby enriching for polynucleotides of said firstlibrary which encode a first immunoglobulin subunit polypeptide which,as part of an immunoglobulin molecule, or antigen-specific fragmentthereof, specifically binds said antigen.
 68. The method of claim 64,wherein said antigen is attached to a substrate selected from the groupconsisting of a synthetic particle, a polymer, a magnetic bead, and aprotein-coated tissue culture plate.
 69. The method of claim 64, whereinsaid antigen is expressed on the surface of an antigen-expressingpresenting cell, wherein said antigen-expressing presenting cell isconstructed by transfecting an antigen-free presenting cell with apolynucleotide which operably encodes said antigen.
 70. The method ofclaim 69, wherein said antigen-expressing presenting cell is constructedin an antigen-free presenting cell selected from the group consisting ofan L cell, a Cos7 cell, a 293 cell, a HeLa cell, and an NIH 3T3 cell.71. The method of claim 65, wherein said antigen is conjugated to afluorescent tag, and wherein host cell pools expressing immunoglobulinmolecules which bind antigen are identified through fluorescenceactivated cell sorting.
 72. The method of claim 53, wherein said effectis antigen-specific binding.
 73. The method of claim 72, comprising: (a)contacting pools of said host cells with said antigen under conditionswherein antigen-specific immunoglobulin molecules expressed by said hostcells will bind to said antigen; and (b) recovering polynucleotides ofsaid second library from those host cell pools, or from replicate poolsof polynucleotides set aside previously, expressing immunoglobulinmolecules to which said antigen was bound.
 74. The method of claim 73,further comprising: (c) dividing said recovered polynucleotides into aplurality of sub-pools and introducing said sub-pools into populationsof host cells capable of expressing said immunoglobulin molecule; (d)permitting expression of immunoglobulin molecules, or antigen-specificfragments thereof, from said host cells; (e) contacting said pools withsaid antigen under conditions wherein antigen-specific immunoglobulinmolecules expressed by said host cells bind to said antigen; and (f)recovering polynucleotides of said second library from those host cellpools, or from replicate pools of polynucleotides set aside previously,expressing immunoglobulin molecules to which said antigen was bound. 75.The method of claim 74, further comprising repeating steps (c)-(f) oneor more times, thereby enriching for polynucleotides of said firstlibrary which encode a first immunoglobulin subunit polypeptide which,as part of an immunoglobulin molecule, or antigen-specific fragmentthereof, specifically binds said antigen.
 76. The method of claim 72,wherein said antigen is attached to a substrate selected from the groupconsisting of a synthetic particle, a polymer, a magnetic bead, and aprotein-coated tissue culture plate.
 77. The method of claim 72, whereinsaid antigen is expressed on the surface of an antigen-expressingpresenting cell, wherein said antigen-expressing presenting cell isconstructed by transfecting an antigen-free presenting cell with apolynucleotide which operably encodes said antigen.
 78. The method ofclaim 77, wherein said antigen-expressing presenting cell is constructedin an antigen-free presenting cell selected from the group consisting ofan L cell, a Cos7 cell, a 293 cell, a HeLa cell, and an NIH 3T3 cell.79. The method of claim 73, wherein said antigen is conjugated to afluorescent tag, and wherein host cell pools expressing immunoglobulinmolecules which bind antigen are identified through fluorescenceactivated cell sorting.
 80. A kit for the selection of antigen-specificrecombinant immunoglobulins expressed in a eukaryotic host cellcomprising: (a) a first library of polynucleotides encoding, throughoperable association with a transcriptional control region, a pluralityof first immunoglobulin subunit polypeptides, each first immunoglobulinsubunit polypeptide comprising: (i) a first immunoglobulin constantregion selected from the group consisting of a heavy chain constantregion and a light chain constant region, (ii) an immunoglobulinvariable region corresponding to said first constant region, and (iii) asignal peptide capable of directing cell surface expression or secretionof said first immunoglobulin subunit polypeptide, wherein said firstlibrary is constructed in a eukaryotic virus vector; (b) a secondlibrary of polynucleotides encoding, through operable association with atranscriptional control region, a plurality of second immunoglobulinsubunit polypeptides, each comprising: (i) a second immunoglobulinconstant region selected from the group consisting of a heavy chainconstant region and a light chain constant region, wherein said secondimmunoglobulin constant region is not the same as said firstimmunoglobulin constant region, (ii) an immunoglobulin variable regioncorresponding to said second constant region, and (iii) a signal peptidecapable of directing cell surface expression or secretion of said secondimmunoglobulin subunit polypeptide, wherein a said second immunoglobulinsubunit polypeptide is capable of combining with said firstimmunoglobulin subunit polypeptide to form a surface immunoglobulinmolecule, or antigen-specific fragment thereof, and wherein said secondlibrary is constructed in a eukaryotic virus vector; and (c) apopulation of host cells capable of expressing said immunoglobulinmolecules; wherein said first and second libraries are provided both asinfectious virus particles and as inactivated virus particles, andwherein said inactivated virus particles infect said host cells andallow expression of said first and second immunoglobulin subunitpolypeptides, but do not undergo virus replication; and whereinantigen-specific immunoglobulin molecules expressed by said host cellsare selected through interaction with an antigen.
 81. An antibody, orantigen-specific fragment thereof, produced by the method of claim 1.82. A composition comprising the antibody of claim 81, and apharmaceutically acceptable carrier.
 83. A method of selectingpolynucleotides which encode a single-domain antigen-specificimmunoglobulin molecule, or antigen-specific fragment thereof,comprising: (a) introducing into a population of eukaryotic host cellscapable of expressing said immunoglobulin molecule a library ofpolynucleotides encoding, through operable association with atranscriptional control region, a plurality of single-domainimmunoglobulin polypeptides, each immunoglobulin polypeptide comprising:(i) an immunoglobulin heavy chain constant region, (ii) an camelizedimmunoglobulin heavy chain variable region, and (iii) a signal peptidecapable of directing cell surface expression or secretion of saidimmunoglobulin subunit polypeptide; (b) permitting expression ofimmunoglobulin molecules, or antigen-specific fragments thereof, fromsaid host cells; (c) contacting said immunoglobulin molecules with anantigen; and (d) recovering polynucleotides of said library from thosehost cells expressing immunoglobulin molecules which bind said antigen.